Gram Staining Principle And Procedure Pdf

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Gram staining method, the most important procedure in Microbiology, was developed by Danish physician Hans Christian Gram in Gram staining is still the cornerstone of bacterial identification and taxonomic division.

The Gram staining technique is the most important and widely used microbiological differential staining technique.

Staining Methods – Simple Staining, Negative Staining, Gram’s Staining and Acid-Fast Staining

Gram stain is probably one of the most commonly used staining procedures used in the field of microbiology. Gram positive bacteria will typically have a stronger affinity for crystal violet on applying gram's iodine than the gram negative cell wall. Being a mordant, gram's iodine forms a complex with crystal violet in the stain that has attached more tightly to the cell wall of gram positive bacteria than that of the gram negative bacteria. After staining the sample with crystal violet, ethyl alcohol is used to decolorize the sample. It achieves its purpose by dehydrating the peptidoglycan layer by tightening and shrinking it. In doing so, large crystal violet cannot penetrate the tightened layer of peptidoglycan, and hence it is trapped in the cell wall of gram positive bacteria. On the other hand, the outer membrane of the gram negative cells cannot retain the crystal violet iodine complex and hence the color is lost.

Clinical microbiology laboratory plays several important roles in the management of bacterial infections. Isolation, identification of pathogenic microorganisms in cultures and subsequent antimicrobial susceptibility testing always assists in selecting appropriate antimicrobial agent and prevention of unnecessary complications. The most important and primary test to perform directly on some special samples such as cerebrospinal fluid and positive cultures is Gram staining which serves as the most rapid and simplest test to characterize microorganisms. It is therefore highly likely that the information provided by the Gram staining will help to assess the adequacy of preliminary diagnosis and antimicrobial therapy selected after collecting culture specimens and before final identification of the microorganism. In recent reports, the impact of Gram staining results on patient mortality has been documented. On the other hand, there remains the possibility that Gram staining results do not match with the final identification of microorganisms. This would carry a risk leading to inadequate antimicrobial therapy and potentially affecting patients' clinical course and mortality.

Differential Staining Techniques

Show Caption Hide Anthrax gram stain. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. The Gram stain procedure distinguishes between Gram positive and Gram negative groups by coloring these cells red or violet. Gram positive bacteria stain violet due to the presence of a thick layer of peptidoglycan in their cell walls, which retains the crystal violet these cells are stained with. Alternatively, Gram negative bacteria stain red, which is attributed to a thinner peptidoglycan wall, which does not retain the crystal violet during the decoloring process. Gram staining involves three processes: staining with a water-soluble dye called crystal violet, decolorization, and counterstaining, usually with safanin.

In their natural state, most of the cells and microorganisms that we observe under the microscope lack color and contrast. This makes it difficult, if not impossible, to detect important cellular structures and their distinguishing characteristics without artificially treating specimens. We have already alluded to certain techniques involving stains and fluorescent dyes, and in this section we will discuss specific techniques for sample preparation in greater detail. Indeed, numerous methods have been developed to identify specific microbes, cellular structures, DNA sequences, or indicators of infection in tissue samples, under the microscope. Here, we will focus on the most clinically relevant techniques.

Gram Staining : Principle, Procedure, Interpretation and Animation

The Gram staining is one of the most crucial staining techniques in microbiology. It gets its name from the Danish bacteriologist Hans Christian Gram who first introduced it in , mainly to identify organisms causing pneumonia. Often the first test performed, gram staining involves the use of crystal violet or methylene blue as the primary color. The term for organisms that retain the primary color and appear purple-brown under a microscope is Gram-positive organisms. The organisms that do not take up primary stain appear red under a microscope and are Gram-negative organisms.

This test differentiate the bacteria into Gram Positive and Gram Negative Bacteria, which helps in the classification and differentiations of microorganisms. When the bacteria is stained with primary stain Crystal Violet and fixed by the mordant, some of the bacteria are able to retain the primary stain and some are decolorized by alcohol. The cell walls of gram positive bacteria have a thick layer of protein-sugar complexes called peptidoglycan and lipid content is low.

The microscope is a very important tool in microbiology, but there are limitations when it comes to using one to observe cells in general and bacterial cells in particular. Two of the most important concerns are resolution and contrast. Contrast, however, can be improved by either using a different type of optical system, such as phase contrast or a differential interference contrast microscope, or by staining the cells or the background with a chromogenic dye that not only adds contrast, but gives them a color as well.

Gram Staining

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2 Response
  1. Philip G.

    When the bacteria is stained with primary stain Crystal Violet and fixed by the mordant, some of the bacteria are able to retain the primary stain and some are decolorized by alcohol. The cell walls of gram positive bacteria have a thick layer of protein-sugar complexes called peptidoglycan and lipid content is low.

  2. Septimo R.

    Gram stain or Gram staining , also called Gram's method , is a method of staining used to distinguish and classify bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria.

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