The Gram stain is a useful stain for identifying and classifying bacteria. The Gram stain is a differential stain that allows you to classify bacteria as either gram positive or gram negative. This gram stain technique was discovered by Hans Christina Gram in 1884. The gram stain procedure separates all bacteria into one of two groups – into gram-negative bacteria which do not stain purple and into gram-positive cells which does stain purple. Bacteria that decolorize easily are called gram-negative, and those that decolorize slowly and retain a primary stain are called gram-positive.
The gram staining procedure consists of fixing a colony of bacteria onto a slide and then flooding the colony with various chemicals. First, crystal violet dye was dropped onto the bacterial cells, staining gram-positive cells purple. Iodine was added to fix the violet dye into place and then ethanol was used to wash the dye off the unstained cells. Finally, a red dye called safranin was used to stain any gram-negative cells present so that they were visible. Once the gram stain procedure was complete, the gram-positive bacteria appeared purple under a microscope while gram-negative cells appear pink or red.
The purpose of this lab was to differentiate bacteria based on the cell wall and to differentiate whether the bacteria was gram-positive or gram-negative. Gram-negative bacteria do not retain the violet dye and are colored red or pink. “Compared with Gram-positive bacteria, Gram-negative bacteria are more resistant against antibodies because of their impenetrable cell wall. These bacteria have a wide variety of applications ranging from medical treatment to industrial use and Swiss cheese production” (2). Materials:
The materials used in the lab were four different gram-staining reagents: crystal violet, Gram’s iodine, 70% ethanol and safranin. Also, a wash bottle of distilled water was used and three glass slides, a petri dish, inoculation loop, Bunsen burner, clothes pins, slide rack, immersion oil and a microscope. The cultures used were Staphylococcus epidermidis, Escherichia coli, and Bacillus subtilis. Methods: First, I put on my safety goggles, put on my apron and put on gloves. I then wipes down the whole area with 70% ethanol and sterilized the table.
To complete the gram-stain, first I prepared and fixed the Staphylococcus epidermidis smear; I cleaned the slides well with distilled water and wiped them off thoroughly. I then labeled each slide as to which culture it was. I then prepared the gram stain of one smear; I made sure to sterilize the inoculation loop by putting it through the Bunsen burner flame until it was red and then let it cool. I added the bacteria on the loop into the water drop on the slide, mixing it thoroughly, and spreading it out into a large, thin layer.
I air dried the slide on the warming tray until the water was gone and then heat fixed it by putting a clothes pin on the slide and putting it through the flame for about four seconds. I let it cool for the staining portion. I then sterilized the loop so that there would be no bacteria left on the loop. I covered the smear with crystal violet and left it on for thirty seconds. I then washed the slide off with distilled water from a wash bottle. Next, I covered the smear with Gram’s iodine for 10 seconds and then washed off the iodine by tilting the slide and squirting water above the smear so that water ran over the smear.
After I decolorized the smear with 95% ethanol and let it stay on for about 15 seconds. I then put safranin on the smear for thirty seconds, and then washed the slide with distilled water and blotted it dry with a clean paper towel. To stain the next slide, I repeated these exact steps but used Escherichia coli as my bacteria culture, I made sure to sterilize the inoculation loop by putting in through the Bunsen burner flame until the inoculation loop turned red, and repeated the same steps just how I did for the first smear. Results:
From doing the Gram stain, I found that the bacteria Staphylococcus epidermidis came out to be gram-positive and Escherichia coli came out to be gram-negative. I viewed Staphylococcus epidermidis under 100x and saw light purple clusters which is exactly what gram positive bacteria is supposed to look like. I viewed Escherichia coli under 100x and saw light gray/ faint pink, thin rods. When doing each step and adding the stains I got the results that were expected. The table below shows the results that were expected I would receive. When staining with crystal violet, both stains came out purple.
When I stained the culture again with Iodine, both cultures came out purple. When I stained the gram positive stain with 95% ethanol, it came out purple and the negative stain came out colorless. Once I used the safranin, the gram-positive came out purple; it was so purple where it was almost a shade of blue. The gram-negative stain came out a faint reddish color. The bacterial culture that came out gram positive was Staphylococcus epidermidis and the bacterial culture that was gram negative was Escherichia coli. Staphylococcus epidermidis was the largest organism and Escherichia coli was the smallest.
I did not agree with the information from my text book, Escherichia coli did not agree with the information because our bacteria smear was under de-colorization. When viewing it under the microscope, I saw light gray clusters with a very light tint of pink instead of bright pink clusters. Gram positive cells more than 24 hours old stain gram negative because the cell wall cannot retain primary stains, also iodine cannot be added before the primary stain in the gram stain. In the gram stain experiment, the only step than can be omitted without affecting determination of the gram reaction is adding safranin.
If I performed a gram stain on a sample from a pure culture of bacteria and observed a field of red and purple cocci, and the adjacent cells were not all the same color, I was conclude that the culture was old. If I viewed a gram-stained field of red rods and purple cocci through the microscope, I would conclude that is a mixed culture. A physician might perform a gram stain on a sample before prescribing an antibiotic to determine its sensitivity to the antibiotic that correlates with the cell wall type. If I were to perform a gram stain on a human cell, the primary stain would be removed easily because human cells do not have cell walls.
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