Gram stain: principle, procedure and results (2024)

Gram stain is a fundamental technique in microbiology, crucial for differentiating between two main groups of bacteria based on the properties of their cell wall. Developed by Hans Christian Gram in 1882, this staining method has become a standardized protocol used in laboratories around the world. In this comprehensive guide, we delve into the intricate details of the Gram stain protocol, exploring its principle, procedure, and importance in microbiological diagnosis.

What is a Gram stain?

The Gram stain is a laboratory test performed to identify bacteria present in samples obtained from suspected sites of infection or body fluids. It involves applying a series of dyes to the sample, leading to the visualization of bacteria under a microscope. The staining process helps classify bacteria as Gram positive or Gram negative based on their reaction to the stain.

Principle of Gram stain:

The Gram stain takes advantage of the differences in the structure and composition of bacterial cell walls to classify them into two different groups: Gram positive and Gram negative.

• Gram positive bacteria: These bacteria possess a thick layer of peptidoglycan on their cell walls, which retains the crystal violet dye during staining, imparting a purple-blue color.

• Gram-negative bacteria: In contrast, Gram-negative bacteria have a thinner peptidoglycan layer and an outer lipid membrane. During staining, they lose the crystal violet dye, but stain with a red dye, appearing pink or red under the microscope.

Gram stain procedure

Gram stain involves a series of steps:

1. Sample preparation:

• Apply a small amount of the sample, obtained from the suspected site of infection or body fluid, to a clean microscope slide.
• Heat the slide by passing it through the flame of a Bunsen burner several times. This process ensures that the sample adheres firmly to the slide.

2. Staining process:

to. Violet Crystal Application:

• Flood the heat-set sample with crystal violet solution, covering the entire slide.
• Let the crystal violet sit for a set amount of time, usually about a minute.

b. Addition of Gram's iodine solution:

• Apply Gram's iodine solution to the slide, completely covering the stained sample.
• Iodine forms a complex with crystal violet, improving its adhesion to the bacterial cell wall.

c. Decolorization:

• Gradually add the destaining agent (e.g., ethanol or acetone) to the slide, drop by drop.
• This step is critical and requires careful monitoring. Excessive discoloration can cause false negative results for Gram positive bacteria.

d. Counterstaining with Safranin:

• After destaining, wash the slide with water to remove excess destainer.
• Apply safranin solution to the slide, covering the entire sample.
• Safranin serves as a counterstain, imparting a contrasting red color to Gram-negative bacteria.

3. Microscopic examination:

• The slide should be examined under a microscope, initially using the X40 objective to evaluate the distribution of the smear. Then examine them using the X100 oil immersion objective.
• All areas of the slide require initial examination. Areas that are only one cell thick should be examined. Thick areas on slides often give variable and incorrect results.
• White blood cells and macrophages stain gram-negative, while squamous epithelial cells stain gram-positive.

Interpretation results:

Interpretation of the results is crucial to determine the presence and type of bacteria in the sample.

• Gram positive bacteria: Retain the crystal violet dye, which appears purple or blue under the microscope.
• Gram-negative bacteria: They lose the crystal violet dye but are stained with safranin and appear pink or red.

Factors that interfere with Gram stain:

Gram stain, being a sensitive procedure, can be affected by several factors:

1. Collection of specimens: Proper sterile collection is crucial to prevent contamination. Previous use of antibiotics can hinder the growth of the organism.
2. Low power analysis (10X):

• The background should appear gram-negative or clear.
  • White blood cells must stain gram-negative; its gram-positive staining may indicate errors.
  • Differentiating fine crystal violet from bacteria is crucial.
  • Smears should be uniform and one cell thick without overlap.

• Note the relative number of cells, such as leukocytes and epithelial cells.
• Observe the arrangements and shapes of the bacteria for clues to their identification.

• Identify and document bacterial morphology, including shape, ends, sides, and axis.
  • Note the pleomorphism, branches or extensions.

Adherence to these guidelines ensures accurate interpretation of the Gram stain, which aids in accurate diagnosis and treatment of bacterial infections.

Clinical significance:

Gram stain aids in the diagnosis of bacterial infections and guides treatment decisions by providing quick preliminary results on the type of bacteria present.

Conclusion:

The Gram stain remains an invaluable tool in microbiology, allowing rapid and reliable differentiation of bacterial types. By understanding the principle, procedure, and interpretation of results, healthcare providers can make informed decisions about patient care and treatment strategies in cases of suspected bacterial infections.

References:

1. Leopold JA, Ferraro MJ. (2020). Concepts and applications of microbiology. McGraw-Hill Education.
2. Forbes BA, Sahm DF, Weissfeld AS. (2007). Bailey & Scott Diagnostic Microbiology. Mosby Elsevier.
3. Tortora GJ, Funke BR, Case CL. (2017). Microbiology: an introduction. Pearson Education.
4. Wilson B. (2017). Staining procedures. In: Wilson B, editor. Techniques in microbiology: a manual for students. Cengage Learning.
5. American Society for Microbiology. (2005). Basic protocol 1: Gram stain of bacterial cultures. In: Walker JM, editor. The Protein Protocols Manual. Human Press.

Biochemical tests for the identification of Gram negative bacteria (thesciencenotes.com)