The Gram stain is one of the most important staining procedures used in microbiology. It is a differential staining technique that involves using crystal violet and safranin dyes to distinguish between Gram positive and Gram negative bacteria. Based on their cell wall structure, bacteria will retain one dye or the other, allowing them to be categorized. Knowing which color bacteria stain can provide valuable information about their identity and properties. This article will review the Gram staining procedure and outline what color Gram positive bacteria appear after staining.
Overview of Gram Staining
The Gram stain process involves applying a series of dyes to a bacterial smear on a microscope slide. Here are the steps:
- A primary stain, crystal violet, is applied to the smear and allowed to soak in for 1 minute. This purple dye penetrates all bacterial cell walls.
- An iodine solution is added which acts as a mordant to bind and fix the crystal violet to the cell.
- A decolorizing agent, usually ethanol or acetone, is then used to wash away excess stain. In Gram positive bacteria, the crystal violet-iodine complex remains trapped. In Gram negatives, the outer membrane is permeable to the decolorizer which washes away the primary stain.
- Finally, a counterstain of safranin is applied which will stain any bacteria that have been decolorized pink or red.
This technique divides bacteria into two large groups based on the physical properties of their cell envelopes. Gram positive organisms have a thick layer of peptidoglycan in their cell wall which retains the crystal violet stain. Gram negatives have a thinner peptidoglycan layer and an outer membrane that does not retain the primary stain when decolorized.
Gram Positive Bacteria Color
So what color do Gram positive bacteria stain? Gram positive bacteria will appear purple or blue-purple after the Gram staining procedure.
The thick peptidoglycan layer of Gram positive cell walls readily absorbs and retains the crystal violet-iodine complex even after decolorizing. Therefore, they will remain purple when viewed under the microscope.
Some examples of Gram positive bacteria that exhibit this purple color include:
- Staphylococcus species like S. aureus
- Streptococcus species like S. pyogenes
- Bacillus species like B. anthracis
- Listeria monocytogenes
- Clostridium species like C. perfringens
- Mycobacterium tuberculosis
- Corynebacterium diphtheriae
Gram Positive Cell Wall Structure
To understand why Gram positives retain the crystal violet, it’s important to take a closer look at the structure of their cell walls:
- The cell wall is very thick, accounting for up to 90% of the cell envelope.
- It is composed primarily of a thick peptidoglycan layer made up of sugars and amino acids.
- Teichoic acids are also present in the cell wall which have a negative charge and may facilitate binding of the positively charged crystal violet dye.
- There is no outer membrane present.
This rigid, thick peptidoglycan layer provides protection for the bacteria but also has small pores that allow substances like the primary stain to penetrate initially. However, once the crystal violet-iodine complex becomes trapped in the peptidoglycan matrix, it cannot be easily removed by decolorizing agents.
Gram Negative Bacteria Color
In contrast to Gram positives, Gram negative bacteria appear red or pink after the Gram stain procedure.
The thinner peptidoglycan layer of Gram negatives allows the decolorizer to easily extract the crystal violet-iodine complex. When the counterstain safranin is subsequently applied, it stains the Gram negatives pink.
Some examples of Gram negative bacteria that exhibit this pink color include:
- Escherichia coli
- Salmonella species
- Shigella species
- Yersinia pestis
- Vibrio cholerae
- Pseudomonas aeruginosa
- Helicobacter pylori
- Neisseria gonorrhoeae
Gram Negative Cell Wall Structure
Gram negative bacteria have distinct cell envelope characteristics that facilitate loss of the primary stain:
- The peptidoglycan layer is very thin, making up only 5-10% of the envelope.
- An outer membrane containing lipopolysaccharide is present exterior to the thin peptidoglycan layer.
- Porin proteins in this outer membrane allow passage of decolorizing agents through the cell wall.
Together, these properties allow the decolorizer to easily penetrate and extract the crystal violet-iodine complex that initially penetrated the peptidoglycan. Only the pink counterstain remains visible when Gram negatives are viewed microscopically after Gram staining.
Clinical Relevance of Gram Staining
The Gram stain procedure is performed frequently in clinical settings because it provides key information about bacteria that can guide treatment:
- Gram positive vs negative classification gives clues about antibiotic sensitivity and resistance. Gram negatives are typically more resistant.
- It narrows down bacterial identity since certain organisms are always Gram positive or negative.
- It gives information about shape and arrangement that provides more identification details.
- When performed on cultures, it provides a preliminary diagnostic test to target therapy early in infection.
Healthcare providers particularly rely on Gram stain results when selecting initial antibiotic therapy for critically ill patients with suspected bacterial infections. Knowing just the Gram classification can help guide choice of empiric antibiotics while awaiting culture and sensitivity results.
Other Differential Stains
While the Gram stain is one of the most useful and widespread, there are a variety of other differential stains that can provide valuable information about bacteria:
Acid-Fast Stain
- Used primarily to identify Mycobacterium species like M. tuberculosis.
- Retains red carbolfuchsin stain after decolorization with acid-alcohol.
- Makes mycobacteria visible as red rods against blue background.
Capsule Stain
- Uses dyes like nigrosin or India ink to visualize capsules as clear halos surrounding stained bacteria.
- Detects encapsulated organisms like Klebsiella pneumoniae, Streptococcus pneumoniae, and Bacillus anthracis.
Spore Stain
- Employs malachite green to stain endospores followed by safranin counterstain.
- Spores appear green against pink vegetative bacteria.
- Identifies spore-forming genera like Bacillus and Clostridium.
Flagella Stain
- Uses dyes like basic fuchsin to directly visualize bacterial flagella under microscope.
- Determines motility and identity of organisms like Salmonella and Proteus species.
These differential stains provide additional identification information beyond the Gram stain to pinpoint bacterial isolates.
Gram Stain Quiz
Let’s review what you’ve learned about Gram staining with a quick quiz:
| Question | Answer |
|---|---|
| What is the primary stain used in the Gram stain procedure? | Crystal violet |
| What color do Gram negative bacteria appear after staining? | Pink or red |
| Which bacteria have a thick peptidoglycan layer in their cell wall? | Gram positives |
| An outer membrane is characteristic of which type of bacteria? | Gram negatives |
| The Gram stain can provide information about bacterial antibiotic resistance. True or false? | True |
How did you do? Understanding the principles behind the Gram stain procedure and the resulting colors for different bacterial types is critical knowledge for microbiology students and healthcare professionals alike. With some review, the main points should stick!
Conclusion
In summary, Gram positive bacteria stain purple or blue-purple after the Gram staining protocol. Their thick peptidoglycan layer retains the crystal violet-iodine complex even after decolorization. In contrast, Gram negative bacteria lose the primary stain and pick up the pink counterstain safranin, appearing red or pink. This differential staining technique provides key information about bacterial cell envelope properties that impacts identification, antibiotic resistance, and infectivity. Being able to correctly describe the color Gram positive bacteria display after Gram staining is essential microbiology knowledge. Consistent review of the Gram staining procedure, cell wall differences between Gram positive and negative bacteria, and the resulting colors will help solidify this vital concept.