Stain And Identify The Bacteria with TM Media's Gram Staining Kit

 

Bacteria are unicellular microorganisms. They have the simplest cell structure. Their genetic information is contained in a single loop of DNA, and some of the bacteria have an extrachromosomal material called a plasmid. The plasmid contains some advantageous genes, such as a resistant gene. Bacteria exist in many shapes, including rod, spherical, spiral, comma, and corkscrew.

Bacteria are ubiquitous in nature; some are harmful, and some are beneficial. To study and understand these bacteria, it is necessary to isolate and identify them. Also, identification is critical in the industries like fermentation industry to improve and develop products, as well as in clinical settings, for early detection of pathogen.

Bacteria are classified into two groups, Gram-positive bacteria, and Gram-negative bacteria, according to their cell wall composition.

Gram Staining



Gram staining is one of the widely used procedures used to differentiate bacteria on the basis of their cell walls' composition.

The technique was devised by Hans Christian Gram in late 1884 when he was searching for a method to visualize the cocci in tissue sections of the lungs of pneumonia patients. The gram staining technique gives information about the phenotypic characteristics of bacteria, or on the basis of cell wall composition, it differentiates bacteria into gram-positive and gram-negative bacteria. Gram-positive bacterial cells have a thick peptidoglycan layer (90% of the wall), whereas Gram-negative bacterial cells have a thin peptidoglycan layer (10% of the wall) with lipid content.

Crystal violet dissociates in aqueous solutions into CV+ and Cl- ions, which pass through the membrane and cell wall of both gram-positive and gram-negative bacterial cells. By interacting with the negatively charged parts of bacterial cells, the CV+ causes the cells to turn purple. When iodine is added, it interacts with CV+ to form large CV-I complexes in the cytoplasm and outer layers of the bacterial cell. The decolorizing agent (alcohol or acetone) interacts with the lipids in the membranes of gram-positive and gram-negative bacteria. Gram-negative cell walls become leaky when exposed to ethanol, allowing large CV-I complexes to be washed from the cell, while the addition of ethanol dehydrates the gram-positive cell's highly cross-linked and multi-layered peptidoglycan layer. The multi-layered nature of the peptidoglycan, combined with the dehydration caused by the ethanol treatment, traps the large CV-I complexes within the cell. The gram-positive cell retains its purple color after decolorization, whereas the gram-negative cell loses its purple color and is only revealed when the counterstain, the positively charged dye safranin, is added. That’s why, at the completion of the Gram staining procedure, the gram-positive cell is purple and the gram-negative cell is pink to red.

Procedure for Gram Staining Technique

The gram staining procedure differentiates bacteria through a series of staining and decolorization steps. At TM Media, we provide a ready-to-use Gram Staining kit for identification purposes. Following are the steps to follow for Gram staining.

 

1.      Using the heat fixation method, fix the sample onto the slide.

2.      Stain the sample on the slide with the primary stain, crystal violet for 1 minute followed by washing the excess stain under a gentle stream of water.

3.      Then, treat the slide with a mordant such as iodine for 1 minute and then followed by a decolorizer for 15 seconds.

4.      Once the treatment of decolorizer is completes, wash off the excess decolorizer under a gentle stream of water. Then treat the slide with counter stain, safranin for 30 seconds to 1 minute followed by washing the excess stain under a gentle stream of water.

5.      Wash the slide in a gentle, indirect stream of tap water and then blot dry with absorbent paper. And once dried, observe under 100x Microscope.

The gram-positive microorganisms will appear bluish-purple in color, and the gram-negative microorganisms will appear pinkish-red.

TM Media, the strategic business unit of Titan Biotech Ltd., offers ready-made staining kits for different types of staining. Their ready-made staining kit collection includes Albert's Metachromatic Stain Kit, Capsule Stain Kit, Field’s Stain Kit, Papanicolaou’s Stain Kit, Schaeffer & Fulton’s Spore Stain Kit, ZN Acid Fast Stain Kit etc.

Along with Staining Kits, TM Media also offers Staining and Indicator Solutions, Analytical and Standard Solutions, and more microbiology lab consumables.

For more, visit https://www.tmmedia.in/

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