Author: Abbas Khan

Abstract:

Gram staining in microbiology is one of the oldest techniques used by Danish Bacteriologist for the first time in 1882 to identify organisms casing pneumonia. Despite the molecular advancement, it is still the most prominent staining technique used in microbiology to differentiate bacteria. It is also used in differentiating other organisms like fungi and parasites. In gram positive bacteria, because of its thick peptidoglycan layer, the stain remains purple/red. Whereas, in gram negative bacteria because of thin peptidoglycan layer, the bacteria under microscope remains pink. Different dyes and reagents are used in this process, firstly, smear is formed on slide with specific culture put through loop and then air-dry or dry heat to remove extra water. After that, the first one involves crystal violet dye. The next one is gram’s iodine which works as mordant. Next is secondary dye known as decolorizer, it is important step in gram staining as it can wash out all the stain if exposed to more tap water. In last steps, safranin is used for counterstaining and then washed out. These culture slides are put forward to microscope to observe not only the color of bacteria but also the shape and morphology.

Introduction:

The name gram staining was first introduced in 1882 by Danish bacteriologist Hans Christian Gram, mainly to identify species causing pneumonia. It is one of the important staining techniques used in microbiology. Gram staining is vital technique used in microbiology to differentiate bacteria into two basic varieties of cells and thus making it fruitful for the initial classification of unknown isolates. That’s why it is considered as primary taxonomic tool. Despite molecular advances in recent past in probes, genera and species, Gram staining is still first priority for identification. As the stain not only retain the shape and form of bacteria but also easy way to determine overall structure of the cells.

The staining differentiates between bacteria by coloring it either pink (gram negative) or purple (gram positive). Gram staining technique can also be used for numerous fungi and other parasites. Gram staining majorly involves four main stages; staining with crystal violet, gram iodine a mordant, decolorization with acetone, and counterstaining with Safranin or Carbol Fuchsin. The differentiation of color is because of the cell wall of bacteria which is either thick (gram positive) or thin (gram negative).

Differential stain methods:

In order to gain more information about micro-organism more accurately, different stain methods are used. This type of method is more specified and accurate than simple methods used in micro biology. These methods involve staining microbes through dyes, in order to get more specified picture of cell and its morphology .

Different types of differential techniques are used in microbiology which includes; acid-fast staining, endospore staining, capsule staining, flagella staining, and gram staining. Among all these the most important and one of the oldest is gram staining, which not only shows the color of the microbes but also their morphology and shape.

Principle of Gram staining:

Gram staining is based on the basis of differences in cell wall and composition of bacteria. Gram positive bacteria which have thick cell wall of peptidoglycan show violet or purple as it resists to decolorization of primary stain. On the other hand, bacteria having thin cell wall of peptidoglycan show pink or red color as it gains counter stain due to less cross link which loses primary stain during decolorization.

Crystal violet dye in aqueous solution dissociate into CV+ and CV- ions. When these ions pass through the cell wall of positive and negative bacteria, the CV+ ions interact with negatively charged component of cell wall and forms purple stain cell. After that Gram’s iodine is added, which works as mordant and interacts with CV+ to produce large complexes within the cytoplasm and outmost layers of the cell. In next step, a decolorizing agent (ethanol or acetone) in aquoues form is added which interacts with lipids in cell wall. The peptidoglycan layer exposes due to dissolvement of outer biological membrane of the Gram-negative bacterial cell wall. The peptidoglycan layer becomes leaky due to thin less cross-linking in Gram-negative cell wall. As a result, loses most of CVI complexes and results in pink color .

In Gram positive bacteria, when decolorizer is added to highly cross-linked and poly layer peptidoglycan, it is dehydrated thus trapping crystal violet and iodine complexes. Purple color remains in Gram positive bacteria because of poly-layer and pink color in Gram negative bacteria as it takes the color of counter stain, safranin.

Sample collection:

Numerous clinical samples/specimens can be used in Gram staining. The collections must be kept in sterile containers. These commonly used specimens are sputum, blood, cerebrospinal fluid, pleural fluid, urine, nostrils, throat, wound etc.

Procedure

Equipment needed:

• Bunsen’s Burner
• Gloves and masks for protection
• Sample display (from body fluids etc)
• Microscopic glass slide
• Sterilize inoculation loop
• Microscope
• Slide rack

Reagents needed:

• Crystal violet dye (known as primary dye)
• Gram’s iodine solution (mordant)
• Acetone or ethanol (decolorizer)
• Safranin (the counterstain)
• Water

Procedure of smear formation:

Firstly, microscopic slides must be contamination free and oil free and should be labelled according to researcher way/procedures. In case of broth, the inoculated loop must be sterile and cooled, put loop full of broth on slide and move it on circular motion. In case of petri dish, sterile water or saline placed on slide and then select the colony which needs to be stained. Inoculated loop is used to pass the require culture to the slide.

The culture is circularly moved across the slide with 15mm in diameter. After that, dry-heat or air-dried is provided to the slide, over heating is avoided, the heat helps in adhesion of cell to the glass slide and prevents huge loss of bacterial culture during rinsing process.

Staining procedure steps:

• The resultant smear is then put on staining rack near the sink and add crystal Violet i.e. the primary stain for almost one minute.
• After that, wash the smear with the help of tap water and add mordant Gram’s iodine for one minute. This step is also known as “fixing the dye”. After that the iodine solution is wash out and extra water is shaken off.
• Next step is “solvent treatment” a few numbers of drop of decolorizer are added to the cultural slide. These decolorizers are mixed solvents of ethanol and acetone. 5 seconds are required for rinsing the smear.
• Unfair staining techniques can lead to incorrect result. In gram staining one of the important step is decolorizing, because it removes outer cell member plus the thin peptidoglycan layer. As the layer is sensitive to alcohols, the layer along with crystal violet dissolves.
• Rinse the water, this stops decolorization process.
• In last steps, add safranin known as counterstain, a few drops of this dye are settled for 60 seconds. This dye either results in pink/red color.
• Last step involves rinsing the water again with tap water.
• Use microscope to observe each bacteria colony, which will be colored pink/purple and also their morphology and shape.

Conclusion:

Gram staining is one of the oldest techniques used to differentiate bacterial cells. This procedure provides important information regarding bacterial cell wall structure and classifications of micro-organisms. These organisms can be divided into two types; gram positive and gram negative, based on their color and dye retaining capability. Different dyes and reagents used in this process. Gram staining is vital in microbiology as it is used for characterization and identification of bacterial species.

References:

1. https://www.ncbi.nlm.nih.gov/books/NBK562156/
2. https://www.tandfonline.com/doi/abs/10.1080/bih.76.3.111.118
3. https://www.researchgate.net/publication/373979703_Gram_Staining_A_Brief_Review
4. https://microbenotes.com/gram-stain-principle-reagents-procedure-and-result-interpretation/
5. https://www.labce.com/spg603164_preparation_of_a_gram_stained_smear_from_culture.aspx
6. https://bio.libretexts.org/Courses/North_Carolina_State_University/MB352_General_Microbiology_Laboratory_2021_(Lee)/04%3A_Staining_Techniques/4.03%3A_Lab_Procedures-_Bacterial_Smear_Simple_and_Gram_Staining
7. https://bio.libretexts.org/Courses/North_Carolina_State_University/MB352_General_Microbiology_Laboratory_2021_(Lee)/04%3A_Staining_Techniques/4.01%3A_Introduction_to_Staining#:~:text=One%20type%20of%20staining%20procedure,%2C%20safranin%2C%20and%20methylene%20blue.
8. https://milnepublishing.geneseo.edu/suny-microbiology-lab/chapter/differential-staining-techniques/
9. https://www.cliffsnotes.com/study-guides/biology/microbiology/microscopy/staining-techniques