The Role of Gram Stain in the Identification of Bacteria

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Determination of the shape and Gram stain of the bacteria under a microscope


            The experiment purpose is to investigate gram stain and shape of bacteria using a microscope. The significance of the grain stain is to indicate whether the bacteria are pink colored (Gram negative) or purpled colored (Gram positive) or both. As asserted by Bauman (14), description of bacteria is usually in form of ‘Gram positive bacillus’ as well as ‘Gram negative cocci.’ Most of the bacteria are transparent, therefore, staining is necessary.


            Grain stain allows for the identification of variation between organisms as well as the variation within an organism.  


 Oil immersion Microscope and microscope Slides

Soap, 95% Ethanol, latex Gloves, Matches clothes, test tube holder, and Bunsen burner.               

Stains:  Iodine gram, Gram’s Crystal Violet, Gram’s Safrani and Acetone/Ethanol Rinse


            Throughout the experiment, Aseptic technique was applied.  Deionized water drop was first placed on a microscope slide and then followed by placement of unknown number 20. Gram staining then began after the heat fixing. Methylene blue or Crystal Violet was then placed where bacteria were heat fixed.  The dye was then rinsed with deionized water and blotted dry after 1min. Mordant Iodine was then placed for another 90 seconds. After the 90 seconds, the blotted dry and deionized water was then used to rinse the slide. 95% ethanol was then used to clean gram negative bacteria. The first non-colored solution drop which slides off the slide was the indication on when to stop using ethanol. After slide rinsing, finally, to counterstain the gram negative bacteria for 60 seconds, saffarin dye was placed on the slide. The unknown number 20 was then viewed by Microscope to determine whether the bacteria were gram negative or gram positive.


            The unknown number 20 that was on a Nutrient Agar slant medium that was found out to be spherical (cocci) appeared to be Gram-negative.

            Discussion and Conclusion

            The gram stain method was applied on a number that was not known. From the result by observation, it appears to be spherical shaped and gram negative. Gram negative bacteria and gram positive bacteria determination is via their variation in cell composition. Gram negative cells could not retain methylene blue iodine complex because of higher content of lipids and peptidoglycan thinner layer in their cell walls unlike gram positive cells which have thicker peptidoglycan (Leboffe et al. 34).

Works Cited

Bauman, R. W. "Microbiology: alternate edition with diseases by body system." (2009).

Leboffe, Michael J., and Burton E. Pierce. Microbiology: laboratory theory and application. Morton Publishing Company, 2012.

August 09, 2023




Subject area:

Bacteria Microbiology

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Expertise Microbiology
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