Exercise number 2 and 3

Exercise 2: Staining Techniques for Prokaryotic Organisms Exercise 3: Subcellular Structure

Khanh Mong Biology Lab 100B Dr. Veno July 24, 2012

Purpose
The general purpose of Exercise 2 is to get familiar with the Gram staining procedure and to differentiate between Gram negative and Gram positive bacteria. The Gram stain is one of the most important staining techniques used today. It is usually performed as the first test in bacterial identification. There are two different groups of bacteria that separated by the Gram technique: Gram-positive bacteria and Gram-negative. It is necessary to identify the two groups because different group require different treatments. In exercise 3, the purpose is to learn the technique of making a wet mount of the living cells. This application of fluorescent techniques allows lab technician to recognize different subcellular organelles. Depends on different characteristics, it would be able to distinguish eukaryotic cells from prokaryotic cells. In eukaryotic cells, the plant and the animal cells could also be differentiated. Also, the exercise requires lab petitioners to stain the smooth cells of rats and view them under a fluorescence microscope.

Materials and Methods

Prokaryotes The materials needed for the practice are microscope slides inoculating loops, Bunsen burners, and slant cultures of Staphylococcus aureus, Escherichia coli, Branhamella catarrhalis, and Bacillus subtilis. A labeling pen and slide holders were needed as well. The process of heat fixing a sample of bacteria was to divide the slides first in half and labeled. Then, we added a drop of water to each side, and then heated the inoculating loop in the Bunsen burner. Next, the cap was taken away from the bacteria and kept at a slant near the flame. After that, the bacteria 2

were mixed with the water on the slide. The sample was smeared around until it became a thin layer, which would be easier to view under the microscope. Finally, four bacteria and the unknown were smeared. The slides were put to air dry, and then they were passed over the flame quickly. The process is to make sure the dryness level, also to kill and heat fix the bacteria. This was done so that the bacteria would not come off with the dye in the staining procedure (MCQUEEN et al. 2012). The materials for Gram stain are: a microscope, slides with heat fixed bacteria dyed with proper procedure, bibulous paper, slide holders, crystal violet, Grams iodine, Acetone-alcohol, Safranin, some markers to mark the slides, and immersion oil to view the slides under 100x. The first stain used was crystal violet, the primary stain. The stain is poured on for thirty seconds and rinsed off with water for three seconds. Then, we put on Grams iodine and left it on for a minute. We also rinse it off with water for three seconds. After rinsing, the slide is held at a forty-five degree angle and a mixture of acetone-alcohol is added for about eight seconds. The slide is rinsed one more time. Finally, safranin is added and left on for a minute. The slide is rinsed one more time and blotted dry with paper. It is then viewed under the microscope. When viewing on 100x, we must use immersion oil (MCQUEEN et al. 2012).

Eukaryotes The materials for exercise 3 are: a clean toothpick, clean microscope slide, methylene blue, and compound scope for examine the cheek cells. One of the group members scraped the inside of his cheek with a toothpick several times. The sample were deposited onto the slide and mixed with a drop of water. One drop of methylene blue was added to apply color to the cheek cells. After locating the cells, higher power is used to observe the structures of the cheek cells. 3

For the fluorescent stain part of the exercise, my group was assigned to do it using Phallotoxins. The materials used for this exercise are rat smooth muscle cells, a fluorescent microscope. First, the cell medium, which contains the rat cells are pipette off. After that, the staining solution is added and held for 30 minutes. The stain is pipette off afterward and the cells are rinsed in 1 mL PBS and 500 uL PBS.

Data Analysis (No data analysis needed for this report) Results

Based on the result of the Gram stain on Staphylococcus aureus, it is Gram-positive because it has a purple color at the end. When we observed the sample under 100x magnification, we could see that the bacteria had a cocci shape. Escherichia coli were Gramnegative as well because they had pink color and rod shape. Branhamella catarrhalis was also Gram-negative since it is also pink. These cells had cocci shape. The Gram-stain made Bacillus subtilis become purple color , so it was Gram-positive. These cells were also rod. At last, our unknown (#3) was Staphylococcus aureus since it has the purple hue and cocci shape. For human cheek cells, the structures we observed were the nucleus, cell membrane, and the cytoplasm. For rat smooth muscle cells exercise, microfilaments are dyed by Phallotoxins. Phallotoxins produced a major lesion which injections are in the liver associated with the stabilization of actinic fiber.

Discussion
1) Addresses HW questions for exercise 2

- If decolorization step is omitted, S. aureus would still be purple. A possible case is that it might adapt to a lighter color of purple because iodine serves to intensify the first color not make it stick. - The bacterial cell structure plays the most important role in determining if an organism is Gram-Positive or Gram-Negative is cell wall, because if it can retain the stain, even after alcohol wash, it is Gram-Positive, other than that it would be Gram-Negative - It is necessary to have a fresh culture when performing a Gram stain because new cultures have the best form of peptidoglycan in their cell wall. Old cultures have a higher chance of turning from Gram-Positive to Gram-Negative thus altering the results of the Gram staining test. 2) Completes Table 3.2 Functions of four organelles: - Endoplasmic reticulum: involved with intracellular transport. - Ribosome: Assembly of amino acids into proteins. - Golgi apparatus: receive materials from the ER and modifies them, synthesize pectin. - Plasma membrane: regulate membranes of materials into a cell and exit of material from a cell. Location of three organelles: - Nucleus: located in the cytoplasm of eukaryotic cells. - Endoplasmic reticulum: located around the nucleus of all cells. - Lysosome: located in cytoplasm of many animal cells. 5

3) + Similarities between prokaryotic & eukaryotic cells: - Enclosed by plasma membranes. - Contain ribosomes. - Have DNA. - Filled with cytoplasm. + Differences between prokaryotic & eukaryotic cells: - Size: Eukaryotic cells are much larger and more complex - Nucleus: Prokaryotic cells do not have a nucleus, while eukaryotic cells do. Supposingly, we are suppose to observe that eukaryotic cells are large enough that they need a nucleus to keep genetic activities close together and increase efficiency. Prokaryotic cells are small enough so that a nucleus is not necessary to maintain efficiency. Instead, prokaryotic cells have something called a nucleoid. The nucleoid is essentially imaginary, and is the region in which genetic activity occurs within the prokaryotic cell. 3) Structure of DNA: Eukaryotic DNA is linear while prokaryotic DNA is circular. Also, Eukaryotic DNA is organized into chromosomes and is complexed with specialized proteins called histones. In Contrast, prokaryotic DNA does not have histones associated with it and prokaryotic DNA does not form chromosomes.

4) Ribosome Structure: Eukaryotic ribosomes are much larger than prokaryotic ribosomes. 5) Organelles: The cytoplasm of prokaryotes does not contain any organelles. Works Cited Berg, Jeremy M., John L. Tymoczko, and Lubert Stryer. "16.1: Glycolysis Is an EnergyConversion Pathway in Many Organisms." Biochemistry. New York: W.H. Freeman, 2002. National Center for Biotechnology Information. Web. 24 Oct. 2010. <http://www.ncbi.nlm.nih.gov/books>. "Cellular Respiration and Fermentation." Symbiosis: The Pearson Custom Library for the Biological Sciences. Ed. Kelly Harris. New York: Pearson Custom, 2009. 53-54. Print. Cummings, Richard D., Jeffrey D. Esko, Hudson H. Freeze, Gerald W. Hart, and Marilynn E. Etzler. "Saccharomyces Cerevisiae, the Model Yeast." Essentials of Glycobiology. By Ajit Varki. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 2008. National Center for Biotechnology Information. Web. 24 Oct. 2010. <http://www.ncbi.nlm.nih.gov/books>. Freeman, Scott. "An Introduction to Carbohydrates, Cellular Respiration and Fermentation." Biological Science. San Fransicso, CA: Benjamin Cummings, 2011. 72+. Print.