Identification of Microorganisms by Differential Staining

Through the process of differential staining, there are distinct differences between the cell walls of gram-positive and gram-negative bacteria. In the case of gram-positive bacteria, the cell wall is comprised of 60-90% peptidoglycan and is very thick. There are numerous layers of teichoic acid bound with peptidoglycan thereby creating very thick cell membranes which causes the cell wall to take up large quantities of basic dye and appears purple. (Hands-on-Labs. (2012)). Conversely, gram-negative bacteria cell walls are much thinner with an outer cell membrane composed of phospholipids and only 10-20% peptidoglycan.

Therefore, it appears pink via the process of differential staining. The difference in color can be attributed to the thinner cell wall and the decolorization process that occurs with the application of the mixture of ethyl alcohol and acetone.

Utilization of Grams mordant solution of iodine on the bacteria generates an insoluble complex, acting as a binding agent. Mordant is defined as a substance, typically an organic oxide that combines with a dye or stain and thereby fixes it in a material (Wikipedia.

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(2013)). When the mordant was applied, a decolorizer (acetone/alcohol) was applied to differentiate the thicker cell walled structures (those that remained purple) from the thinner cell walled structures (those that turned pink).

The differential staining method enabled visualization of the thicker cell walled, gram-positive bacteria Staphylococcus and Lactobacillus, as well as the purple staining of the yeast S. cerevisiae. The color of S. cerevisiae is noteworthy, as it is a fungus. The rules of differential staining do not apply.

The fungus simply picks up the first color/stain utilized, in this case purple (McCarthy, Tom. (2014)). In the differential staining procedure, gram-negative E. coli was visualized and appeared pink.

In the case of gram-positive staining, the Lactobacillus gram positive rods were seen. This bacteria exists in many popular foods including yogurt, cheese, and the fermentation of beer and wine. Alternatively, the bacteria can have negative impact on the human body in the form of infections, commonly involving the urinary tract. Another gram-positive bacteria, Staphylococcus, exists in the nose and on the skin of humans. Found in these locals, it is generally benign, until there is a disruption or injury in the skin that introduces it systemically. (Stoppler, Melissa Conrad MD). Staphylococcus can be insidious and potentially life-threatening if left untreated, causing sepsis if left to run rampant. A yeast delineated as S. cerevisiae appeared purple in this experiment. This would lead one to believe that it is gram-positive as well. However, as a fungus, the rules do not apply, as its cellular structure is different and it takes up whatever color is first introduced. The only traceable source of infection linked to S. cerevisiae is via the use of S. saccromyces as a probiotic during the course of intensive antibiotic treatment and the subsequent proliferation of S. cerevisiae.

Finally, in the case of gram-negative bacteria, E. coli is visualized as pink capsular colonies in the differential staining slides. Utilized as a main fermenting agent with tremendous genetic diversity, it has historically been used in the making of beer, bread and rice wine. (US National Library of Medicine. May 2010)). While E. coli does exist in the human body, introduction of harmful variants through ingestion of contaminated food, for example, can lead to (bloody) diarrhea, dehydration and kidney failure.

References

1. Hands-on-Labs. (2012). A Laboratory Manual of Small-Scale Experiments for the Independent Study of Microbiology. Englewood, CO.
2. Wikipedia – The Free Encyclopedia. en.wikipedia.org. En.wikipedia.org/wiki/mordant.
3. Penn State PHP Service. Gram Staining of Microorganisms. McCarthy, Tom php.scripts.psu.edu/tam5226/?p=gram-stain
4. MedicineNet. Staph Infection: Symptoms and Staphylococcus Aureus Facts. Stoppler, Melissa Conrad MD. www.medicinenet.com/staph_infection/article.htm
5. US National Library of Medicine. E. Coli Infections: Medicine Plus. NIH: National Institute of Allergy and Infectious Diseases
   http://www.nlm.nih.gov/medlineplus/ecoliinfections.html