Lab Report 1 - Grade: C+ - Introduction To Simple Stain And Gram Stain PDF

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Nerixa Portillo BIOL 2310 September 17, 2017 Introduction to Simple Stain and Gram Stain The study of Microbiology is small organisms, but the naked eye cannot see those small organisms. Staining is necessary for Microbiology to distinguish the structure of microbes because they are translucent, light passes through them, and that is why we cannot see it with our own eyes. (Maxwell 2017) There are two major stains they are Simple Stain and Differential Stains, in other words, Gram Stain. There are two categories of stains Acidic and Basic dye. Acidic dye is a negative charge, and Basic dye is a positive charge. The difference between simple stain and gram stain is that simple stain has one simple stain and gram stain have more steps for staining because of the structure of bacteria’s cell wall. The simple stain contains a basic dye which contains methyl blue, crystal violet, safranin, and malachite green. Though acidic dyes do not stain the cells; they stain the background because the cells repel the negative charge dye. The purpose of simple stain is to visualize the structures of bacteria. Gram stain is a complex stain, the reason for gram stain is because cells of different species have different physical and chemical properties. (Maxwell 2017) It is also a used procedure for staining bacteria. Gram Stain is mostly in use in the clinical setting and research laboratories. (Becerra et al., 2016) Gram-Positive bacteria usually appear purple with their thick cell wall, and GramNegative bacteria appear pink with their thin cell wall. (Anderson et al., 2016 pg. 53-54) Procedure for Simple Stain and Gram Stain To stain a bacterium, we should already have comprehended the aseptic technique. Only one bacteria will be in this procedure for simple stain, and that is E. coli. Then transfer the bacteria onto a slide, allow to dry then heat-fix the slide for the bacteria to stay in place. After drying, the bacteria is ready for stain Methylene Blue. Let it sit for one minute then rinse with water and allow it to dry then it will be able to visualize the bacteria; its appearance will be a blue or purple color. For Gram stain, always using the aseptic technique to transfer bacteria. The bacteria will be E. coli, B. subtilis, P. fluorescens, and S. aureus. After it has been dry and heat-fix, it will be

ready for the first stain, which will be the crystal violet setting it for one minute then rinsed off with water. The next step would be applying Gram’s Iodine, which is mordant, it will fix for another one minute then rinsed off. Next, adding the decolorizing agent and that is the alcohol solution, but it will set for 10-12 seconds, then immediately rinsed. The alcohol solution removes Gram’s Iodine from a gram-negative, but not from the gram-positive bacteria. Finally, the last step would be applying the counterstain safranin. For gram-negative bacteria, the cells turn pink and as well gram-positive does not make a difference since they are already purple. Results Simple Stain of Escherichia coli The actual results for E. coli are that it should be clusters of tiny dots. Their structure is a rod-shape, and they are gram-negative. On the picture of E.coli, it is unable to find the cluster of tiny dots, either because it was not heat-fix right or the cells just washed away by not staining it correctly. The reason for this is to view how E. coli is the size and structure.

E. coli stained under 100x oil immersion (Portillo, Nerixa 2017)

Escherichia coli Gram Stain The actual results for Gram staining E. coli after applying safranin the color would appear pink, can picture is as a rod-shape, and since the color is pink, then it would be gram-negative. In this picture for E.coli, it is a lab partner’s bacteria unfortunate the mistake was letting the dye’s stay on too long; we can see the clusters of rodshaped and the color is pink, it will classify as gram-negative.

E. coli gram stain under 100x oil immersion (Argueta, Stephanie 2017)

Pseudomonas fluorescens Gram Stain

The gram stain results for P. fluorescens should stain red/pink because they are gramnegative bacteria, they stain red/pink because they have thin cell walls and they are high fats which mean they do not hold onto the crystal violet stain. Following the steps carefully is very important to remove the dye’s and that was a mistake, not checking time correctly. In this picture for P. fluorescens, it appears to be a dark reddish with tiny marks of pink, with rod shapes. Since it is a pink color, this means it is gram-negative. P. fluorescens under oil immersion 100x (Osbourne, Paige 2017)

Bacilus subtilis Gram Stain For B. subtilis would stain purple, because of their thick layer cell wall that would mean that they are gram-positive and their shape is rod-shaped. The reason the bacteria stays purple because the crystal violet gets stuck on their thick cell wall. What the mistake is making on this gram stain is not smearing the bacteria completely and not being heat fixed correctly. In this picture of B. subtilis, the bacteria seem to be close together with their purple color meaning it is gram-negative. The bacteria is seen clearly and separated.

B. subtilis under 100x oil immersion (Chalabiani Nejadazar, Tina 2017)

Conclusion These results are accurate figuring out if they are stained correctly. Finding their correct shape and charge are exact as the actual results. Simple staining and Gram Stain is necessary because the naked eye is unable to view these microbes, viewing under a microscope is helpful. Comprehending the simple stain and gram stain is huge in Microbiology. The understanding between gram-negative and gram-positive because of their cell walls characteristics like if it is thick or thing. Bacillus does not stain with Gram stain; the reason is that it is an endospore. Since the structure was clear, to correctly stain it, we would apply an endospore stain, which will appear malachite green. (Anderson et al., 2016 pg.b55) A microbiologist can visualize the stained bacteria by its physical and chemical properties of their shape, size, and even their

charged cell wall. In a clinical setting, would be astonishing for staining, because there could be a bacterium infecting humans and by staining we can see the properties of that type of bacteria and figure out how to cure it.

Works Cited Anderson, D. G., Salm, S., Allen, D., & Nester, E. W. (2016). Nesters microbiology: a human perspective. New York, NY: McGraw-Hill Education. Becerra, S. C., Roy, D. C., Sanchez, C. J., Christy, R. J., & Burmeister, D. M. (2016). An optimized staining technique for the detection of Gram positive and Gram-negative bacteria within the tissue. BMC Research Notes, 91-10. doi:10.1186/s13104-016-1902-0 Maxwell, R. (2017). Lab 2: Biological Stains. GSU iCollege. Retrieved on September 17, 2017 from http://gsu.edu/lab2.pdf....