Lab Report

Muna Maharjan

Gram Staining of Escherichia coli and Staphylococcus epidermis

Texas A&M Commerce University

2420 Microbiology Lab

 

 

Abstract

This experiment shows the Gram staining method performed on two bacteria: Escherichia coli and Staphylococcus epidermis. The Gram staining method differentiates bacteria into Gram-positive and Gram-negative by the staining method using primary stain, mordant, decolorizer, and secondary stain. The two smears are obtained from the two colonized streak plates of E. coli and S. epidermis and then heat fixed, then followed the gram staining procedure one after another. The results were as expected, positive for S. epidermis and negative for E. coli. The results could be better if a proper sterilizing technique is used and be more specific with time for dye and decolorizing.

(Keywords: E. coli, S. epidermis, Gram-positive, Gram-negative)

Introduction

The process of identifying the bacteria by staining with basic dye which helps to visualize the bacteria more clearly is known as gram staining. Bacteria were first treated with a primary stain followed by mordant, then decolorizer, and lastly the secondary stain. The thickness of the peptidoglycan layer is determined by the absorbent of the primary dye during the process. The process is named after Hans Christian Gram, who first introduced the method of Gram staining in 1884 while examining the lung tissue from his patient who died of pneumonia. He performed this process first on Streptococcus pneumoniae and Klebsiella pneumoniae. He used Gentian(crystal) violet as a primary stain where he uses Lugol’s solution as a mordant and he used ethanol to rinse away the dye. He saw pneumococci retained the primary dye and others were decolorized by the alcohol and need secondary dye to absorb some color (Hardy,2016). From this, we can say that there are mainly two types of bacteria, Gram-positive and Gram-negative. Escherichia coli (E. coli) is Gram-negative. It is rod-shaped and these bacteria make ATP by aerobic respiration if oxygen is present, so they are also known as a facultative anaerobic bacterium. Although these bacteria can infect the gastrointestinal tract of humans and animals, they are not considered harmful (Lim, 2013). Staphylococcus epidermis (S. epidermis) is a common Gram-positive that can be found commonly on the skin and mucous membranes of humans and other mammals. It is assumed that these bacteria protect human’s and animal’s skin from the harsh conditions in the natural habitat if encountered (Otto, 2009).

Methods and Materials

The working area was properly sterilized with alcohol before starting to avoid contamination. We were given two samples E. coli and S. epidermis. We spread a drop of deionized water on the slides and spread them. We transferred the samples from the streak plate to the slides with the help of an inoculated loop. We sterilize the loop with the blue flame before and after taking the sample from the streak plate. We let the slide air dry before we do the heat fixing method. After the slide cooled from heat fixing, we flood the slide with a primary stain (crystal violet) and waited for a minute. Then we used the iodine solution as a mordant same way and let sit for 45 seconds. And we pour our decolorizer on to the slide. We rinsed the slide with DI water after every step but after the decolorizer, we used it right after. Lastly, we poured safranin, a secondary stain. After a minute we rinsed the slide with DI water again and dried the slide by gently tapping it with the lens cleaning paper.

Result

After viewing the S. epidermis slide on the microscope, we could see the purple stain on it. We viewed the slide under 1000x magnification (oil immersion) and saw the spherical and clustered grape-like structures. The purple stain on the smear indicates S. epidermis is Gram-positive.

Fig II: S. epidermis under 1000x

Fig I: S. epidermis under 400x

The first figure is the image of S. epidermis in 400x magnification and the second is under the oil immersion lens. Similarly, E. coli was also observed under two magnification,400X and 1000X respectively. On the 400x we saw very small pink structures indicating Gram-negative and under 1000x (oil immersion) we saw rod-shaped structure more likely individuals.

Fig I: E.coli under 400x

Fig II: E. coli under 1000x

The first figure is under 400x magnification and the second is under 1000x magnification.

Discussion

The result was obtained as expected Gram positive for S. epidermis and Gram-negative for E. coli. We should be more careful while transferring the colony from the streak plate to the smear to avoid as much contamination as we could. We should perform our process more than one time to avoid any kind of possible error. We should be precise while doing the decolorizing step because we could bleach all our work if not careful. While transferring the colony from streak plate to smear we must let the inoculated loop cool down otherwise we will end up killing our species. If we follow these instructions carefully, we can get better results in the future with a clearer vision of the shape and morphology of the bacteria.

References

Jay, H. (2016). Gram's Serendipitous Stain. Hardydiagnostics.com. Retrieved 4 October 2020, from http://hardydiagnostics.com/wp-content/uploads/2016/05/Hans-Christian-Gram.pdf.

Lim, J. Y., Yoon, J. W., & Hovde, C. J. (2010, January). A Brief Overview of Escherichia coli O157:H7 and Its Plasmid O157. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3645889/

Otto M. (2009). Staphylococcus epidermidis--the 'accidental' pathogen. Nature reviews. Microbiology, 7(8), 555–567. https://doi.org/10.1038/nrmicro2182

Rowan, E. & Karki, M. Gram Staining Procedure Lab Video, Microbiology lab. Lab 5 results. Accessed: 10/03/2020