Table of Contents
Introduction to Gram Staining technique: A Cornerstone in Microbiology
Gram staining is an important technique in microbiology. It is a staining method that narrows down the classification of bacteria into two broad groups: Gram-positive and Gram-negative.
It is named after Hans Christian Gram, a Danish bacteriologist. Bacteria are a large group of microorganisms that cause a wide range of infections, worst, they are ubiquitous. Gram stain became an indispensable tool for microbiologists to make an informed guess as to what kind of organism(s) is responsible for an infection.
Basics for Gram classification
Gram staining classification is based on the structural differences in the cell walls of bacteria, chiefly the peptidoglycan layers. The cell wall of Gram-positive bacteria has a thick peptidoglycan layer (90% of the cell wall). Whereas, the cell wall of Gram-negative bacteria contains a thin peptidoglycan layer (30% of the cell wall).
Principle of Gram staining
The first step in Gram staining is covering the smear with a primary stain crystal violet. Then the stain is fixed with a mordant, Lugol’s iodine, which forms a complex with the primary stain. This will prevent it from washing off easily.
The next step is decolourizing the smear with alcohol. It is a determining step in Gram staining. Decolourization causes the organisms with thin peptidoglycan layers to lose their stain, whereas the organisms with thick peptidoglycan layers, still retain their stains as a result they are termed Gram-positive.
A secondary stain neutral red, is then applied to the smear; to allow the organisms that lost their stain to take up another colour.
Under the microscope, the organisms that appear blue or purple are the Gram-positive organisms, while the ones with pinkish–red colour are the Gram-negative organisms.
Materials Needed for Gram Staining
Reagents:
- Crystal violet (primary stain)
- Lugol’s iodine (mordant)
- Decolourizer (ethanol or acetone)
- Neutral red (counterstain)
- Water
Equipment:
- Microscope Slides
- Bunsen burner
- Microscope
- Immersion oil
Procedure for Gram Staining: A Step-by-Step Guide
1. Preparation of the Smear
According to Monica Cheesebrough, smears should be evenly spread on an area of about 15-20 mm diameter on a grease-free slide. Smears can be made from different specimens; purulent samples (containing pus), non-purulent fluid (sediments after centrifugation), culture, sputum, swabs, stool, and skin smears.
- A small quantity of the specimen is spread thinly on a clean glass slide.
- The smear is allowed to air dry completely.
- The slide is then heat-fixed by passing it through a Bunsen burner flame two or three times to fix the smear to the slide.
2. Application of Crystal Violet
- Crystal violet, a primary stain, is applied to the heat-fixed smear and left to sit for a specific time (usually one minute).
- The slide is then rinsed gently with water to remove excess stain.
3. Iodine Treatment
- Lugol’s iodine, is added to the slide and left to sit for one minute. This step acts as a mordant, forming an insoluble complex with the crystal violet within the bacterial cell wall.
- The slide is rinsed with water.
4. Decolorization
- The slide is decolorized with a mixture of acetone and alcohol. This step is important as it differentiates Gram-positive from Gram-negative bacteria. Gram-positive bacteria retain the crystal violet-iodine complex, while Gram-negative bacteria lose it.
- The slide is immediately rinsed with water to stop the decolorization process.
5. Counterstaining with neutral red
- Neutral red is applied to the decolorized smear and left for 30-60 seconds. This stains the Gram-negative bacteria red.
The slide is rinsed with water, air-dried, and examined under a microscope using Oil immersion lens (x100).
Result report and interpretation.
Gram-Positive Bacteria
They appear purple or blue under the microscope. And are further identified by their shapes and arrangement.
Examples include
Cocci (round)– Staphylococcus spp. Streptococcus spp. enterococcus spp.
Bacilli (rods)– Bacillus spp. Clostridium spp.
Gram-Negative Bacteria
They appear pink or red under the microscope.
Examples include
Cocci (round): Neisseria spp. Moraxella spp. Acinetobacter spp.
Bacilli (rods): Escherichia coli, Salmonella spp. Pseudomonas spp. campylobacter spp.
Limitations of Gram staining
- Gram staining is a preliminary step in bacterial identification. Further tests are often required for definitive species identification.
- Some bacteria may exhibit atypical staining patterns (Gram-variable or Gram-indeterminate).
- The Gram stain does not apply to all microorganisms, such as fungi and mycobacteria.
Therefore, although Gram staining helps the microbiologist to understand the class of organism involved, it’s important to combine it with other microbiological and biochemical identification tests, for accurate species typing.
