Gram Staining and Microscopic Techniques in the Microbial Identification

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Overview of Gram Staining

Gram staining is still an important staining method in bacteriology. It remains the universally accepted first step in the identification of bacteria, including anaerobic bacteria. It categorizes bacteria into two broad groups, Gram-positive and Gram-negative, a distinction based on cell wall composition. Gram staining usually uses crystal violet or methylene blue as the primary color in the first test. Organisms that retain their primary color and appear purple-brown under the microscope are Gram-positive organisms. Microorganisms that do not receive primary staining appear red under the microscope as Gram-negative bacteria.

The Procedure of Gram Staining

  • Preparation of a Slide Smear

Suspension culture droplets are transferred to microscope slides using inoculating loops, requiring only a small amount of culture. The culture medium was evenly diffused with the inoculum ring. Slides can be fixed by air-dried or heated over a gentle flame.

  • Gram Staining

1. Crystal violet stain is added over the fixed culture. After 10-60 seconds, pour out the stain and rinse the excess with water.

2. Iodine solution is used to cover the smear for 10 to 60 seconds. This step is known as "fixing the dye". Wash with water for 30 seconds.

3. A few drops of decolorizer are added to the slide. The decolorizer is usually a mixture of ethanol and acetone. This step is called "solvent treatment". The slide is flushed with water in 5 seconds.

4. The smear is counterstained with a basic fuchsin solution for 40 to 60 seconds. Rinse the fuchsin solution with water and blot the excess water with blotting paper.

Gram-staining procedure.Fig.1 Gram-staining procedure. (Mubarak, 2017)

Microscopic Examination of Slide

Specific morphological details are required to characterize a microorganism, which are usually determined by means of light microscopy. The smear distribution of the section should be examined initially with an X40 objective, followed by an X100 oil immersion objective. Thick areas of the slides tend to give variable and incorrect results.

Gram staining reactions of the bacteria.Fig.2 Gram staining reactions of the bacteria. (Alamu, 2021)

Microscopy-based observations are still often used to define interesting morphological differences in bacteria such as streptococci, staphylococci, and bacillus (e.g., Listeria monocytogenes, E. coli or Salmonella spp.), and Vibrio, in both clinical and research sceneries. Nonetheless, microscopy alone is not sufficient for microorganism identifications for several reasons: small cells that are usually present are difficult to identify; prokaryotes vary widely in size and some cells are close to the resolution limits of the optical microscope; when observing natural samples, such cells can easily be missed, especially if the sample contains a large amount of particulate matter or a large number of larger cells; and it is often difficult to differentiate living cells from dead cells or cells from inanimate materials present in natural samples.

If you are in the initial stage of LBP development, you can completely rely on Creative Biolabs' microbial identification services to help your project advance quickly. Contact us for detailed communication, and we will guide your research throughout the whole process.


  1. Mubarak, Z.; et al. Comparison of Candida sp. Colonies in Gargling-volume Culture from Subject Wearers of Heat-cured and Self-cured Acrylic Resin Removable Partial Dentures. World Journal of Dentistry. 2017,8. 471-474.
  2. Alamu, E.O.; et al. Physicochemical, microbial, and aflatoxin analyses of selected high-quality cassava flour (HQCF) from the major markets of Zambia. Cogent Food & Agriculture. 2021, 7(1): 1914906.

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