Bac:Gram Stain worksheet PDF

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Feb 20, 2024

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Bacterial stains: Gram stain and other differential stains for bacteria Part 1: Gram Staining Bacteria 1. Before comple-ng this virtual lab, make sure to go over any background material (videos, powerpoints, etc) so that you understand the concept and can answer the ques-ons asked. Look over the ques-ons in this sec-on so that you can take some notes, especially on the steps of the gram stain and what is happening at each step. These suppor-ng resources can be found in the Gram Stain lab folder. a. Use the following video link ( LINK ) to answer the following ques-on and to fill in the table below. I have provided the steps/dyes used in the gram stain. Fill in the rest of the table. The module 4 powerpoint may also be useful here. I have filled in the first row. Make sure to include the importance of each step/dye. (This video is also available as a link in the lab folder in Bb). 1) What do we mean when we say the Gram stain is a differen-al stain? A gram stain is able to tell the difference between a specific bacteria. Table: Chemical or dye used What is happening at this step Color of gram posi-ves aSer this step Color of gram nega-ve aSer this step Crystal violet / rinse All cells are stained purple with this dye so that we can see them under the microscope Purple Purple Iodine / rinse Mordant helps with cell wall adherence Purple Purple Ethanol / rinse It washes away stains from cell walls that are gram nega-ve Purple Colorless

b. Why do you think it is important to rinse aSer each step? So that the next step(s) won’t be affected by the previous ones c. Safrinin is called a counter stain. Why is the use of a counterstain important in the Gram stain? Why can’t we just rely on crystal violet? You cannot rely on crystal violet because both gram posi?ve and gram-nega?ve bacteria will appear purple. When you use a counterstain aBer the decolorizer is it will directly stain the bacteria that has been decolorized. That being the gram nega?ve due to the thin cell wall layers of pep?doglycan losing its color from the ethanol d. Brieﬂy describe the difference between the gram posi-ve and gram nega-ve cell wall. Because the Gram stain can dis-nguish between a Gram posi-ve bacteria and a Gram nega-ve bacteria, it is called a differen-al stain. How does the Gram stain dis-nguish between the two types of bacteria? To answer this ques-on, note that the ethanol/ destain step is the key step to differen-a-ng Gram posi-ves from Gram nega-ves. Why do the two types of cells react differently to this step? (see chapter 4 informa-on on the Gram stain, along with videos). The thickness of the pep?doglycan is the difference between a gram posi?ve cell wall and a gram nega?ve cell wall. The posi?ve has a thicker layer of pep?doglycan. You can tell the difference from the stain, they appear two different colors. They each react differently due to the thickness of the pep?doglycan. The iodine is unable to penetrate through the walls of the posi?ve due to the thickness. Safranin / rinse Makes dye able to s-ck to gram nega-ve cell walls Purple Pink Chemical or dye used What is happening at this step Color of gram posi-ves aSer this step Color of gram nega-ve aSer this step

e. In the lecture por-on, we learned that the Mycoplasmas are a group of bacteria that don’t have a cell wall. Go over each step of the Gram stain and look at your answer for ques-on ‘d’ above. Would you expect them to stain Gram posi-ve or Gram nega-ve? Using everything you learned, explain your answer to this ques-on (the explana-on is the most important part). Since they do not have a cell wall containing pep?doglycan, they would not be able to retain the crystal violet. The crystal violet and iodine complex would be removed during the decoloriza?on complex. The decoloriza?on dissolves the lipids in the cell walls of bacteria, making it easier to remove the crystal violet. Part 2: Trouble shooting the Gram stain Your lab instructor has provided you with a culture of E. coli, a Gram nega-ve bacteria. You will be performing the Gram stain on this bacteria. 1. What color and shape/morphology would you expect if your Gram stain turned out perfect. Draw an image of your expected result (use the correct color and shape). Take a screen shot, snippet, or picture with your phone and insert your image here. E. Coli is a gram- nega?ve bacteria. If I did a gram stain with E. coli and another gram- nega?ve bacteria, both bacteria would be stained pink with the safranin. This is because of their thin layer of pep?doglycan. If the other bacteria were Neisseria gonorrhea, we would see a pink stained diplococcus, a round bacteria that come in pairs. We would also see rod shaped single bacilli-stained pink; this would be the E. coli.I would expect the mycoplasmas stain to be gram- nega?ve. 2. Insert image below (or a_ach as a separate file). 3. Use the following video link ( LINK ) to see the Gram stain procedure on your E. coli culture. Answer the following ques-ons. a. List the major steps of the Gram stain, including -mes (hint, the heat fixing step is done no more than 2 seconds). 1. Flood slide with crystal violet for 1 min. 2. Rinse with dis?lled water. 3. Flood slide with iodine for 1 min. 4. Rinse with dis?lled water. 5. Decolorize with ethanol for 5-10 seconds. 6. Rinse with dis?lled water. 7. Flood slide with safranin for 1 min. 8. Rinse with dis?lled water

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b. What were the results of your Gram stain? Make sure to state the Gram stain results along with colony morphology/arrangement of any bacteria present. Use proper terminology from the powerpoint (not rods, spheres, or circles). There was gram- nega?ve and gram- posi?ve bacteria. I could tell by the two different colors of stain. The gram-nega?ve bacteria are stained pink. Streptobacillis moniliformis are gram- nega?ve streptobacilli bacteria because they are in long filament shaped. The gram- posi?ve bacteria is stained purple. The staphylococci are gram-posi?ve cocci bacteria that are arranged in clusters. c. What do these results tell you about your E. coli culture? Is it pure? How do you know? This tells me that the E. coli culture is a mixed culture. It is not pure because there are other organisms in the culture. I know this because E. coli is a gram- nega?ve bacteria that is stained pink. There is evidence of a gram- posi?ve bacteria of different morphology is the culture. d. What would be your next step if you want to work with E. coli (think about the previous lab)? I would have to isolate the E. coli by using the quadrant streak plate. This would dilute the culture un?l there were only a few of each organism leB. ABer the 24- 48 hour incuba?on period, the two bacteria would be isolated into colonies. I would then be able to take a culture from the isolated colony of E. coli. 3. Use the following video link ( LINK ) to watch a Gram stain that has gone wrong. Using your detailed steps in ques-on 2a, what went wrong in this Gram stain? How did it affect your result? Provide an explana-on for your answer. The sample was not fixed by running it trough the Bunsen burner. With no heat, the culture is not fixed. The smear would not be able to decrease the amount of excess liquid and could possibly wash away the bacteria. If this step was missed, you wouldn’t see anything under the microscope. 4. Use the following video link ( LINK ) to watch a Gram stain that has gone wrong. Using your detailed steps in ques-on 2a, what went wrong in this Gram stain? How did it affect your result? Provide an explana-on for your answer. The sample was not fixed by running the slide through the Bunsen burner 2- 3 ?mes for about a second each pass . Without the heat, the culture is not fixed to the slide ul?mately the bacteria not fixed in the slide . And the smear would not be able to decrease the amount of excess liquid and could wash away the bacteria and we would not see anything in this slide under microscope .

5. Use the following video link ( LINK ) to watch a Gram stain that has gone wrong. Using your detailed steps in ques-on 2a, what went wrong in this Gram stain? How did it affect your result? Provide an explana-on for your answer. The slide was overexposed to the heat. Overhea?ng the slide can alter the bacteria's morphology. We would not be able to accurately iden?fy the bacteria under the microscope. It is hard to tell the morphology of the bacteria as well as dis?nguish whether they are gram- nega?ve or gram- posi?ve. Part 3: Acid fast stain, Endospore stain, and Capsule stain 1. Watch the following video ( LINK ) in this folder. You will be answering ques-ons on this video, so please go over the ques-ons first so you know where to take notes. Complete the following ques-ons: a. Why don’t acid fast bacteria stain well with the Gram stain? The crystal violet is unable to penetrate the cell walls of an acid-fast bacteria. b. Name one clinically significant genus of bacteria that is acid fast (the one he discussed mostly in the video)? What are two diseases caused by members of this genus? Mycobacterium, it can use tuberculosis and leprosy. c. What would happen if you did not heat the acid fast stain? There would be no stain pigment because the heat is what pushes it in to the cell walls. d. What is an endospore? Its a tough and non-reproduc?ve dormant by bacteria from the firmicutes and phylum e. How do we drive in the malachite green stain before decoloriza-on? By using heat f. What would happen if you did not do the hea-ng step during the endospore stain. There would be no pigment in the cell walls.

i. Which two genera commonly make endospores? Bacillus and Clostridium g. What is a capsule? What are the func-ons of a capsule (go back to chapter 3 in the lecture textbook!)? I ts a protec?ve layer that’s kinda slimy and allows the cell to adhere to surfaces. The func?on is to prevent the cell from being eaten/ destroyed from white blood cells. h. What is a nega-ve stain? A staining procedure in which the structure isn’t stained i. Now that you have gone over all four stains in parts 1-3, discuss how each one meets the defini-on of a differen-al stain. The gram stain meets the defini?on of differen?al stain by staining gram posi?ve bacteria with crystal violet and gram-nega?ve bacteria with safranin. 2. The acid-fast stain meets the defini?on of differen?al stain by staining acid-fast bacteria with carbon fuchsin and nonacid fast bacteria with methylene blue. 3. The endospore stain meets the defini?on of differen?al stain by staining a vegeta?ve cell with malachite green and an endospore cell with safranin 4. The capsule stain meets the defini?on of differen?al stain by actually being a nega?ve stain with the capsule appearing colorless and the bacteria appearing purple. Part 4: Results interpretation Go over the staining lab powerpoint (also posted) and answer the 10 ques-ons on the slides. 1. The result of the gram stain is all gram-nega-ve bacteria. The cell morphology is bacillus, and the cell arrangement is diplobacilli. 2. The result of the gram stain is all gram-posi-ve bacteria. The cell morphology is bacillus, and the cell arrangement is streptobacilli. 3. The result of the gram stain is all gram-posi-ve bacteria. The cell morphology coccus, and the cell arrangement is diplococci and tetrad. 4. The result of this gram stain is all gram-posi-ve bacteria. The cell morphology coccus, and the cell arrangement streptococci. 5. The result of this gram stain is all gram-posi-ve bacteria. The cell morphology is coccus, and the cell arrangement staphylococci. 6. That is a endospore stain. 7. The arrow is poin-ng to a bacterium with an endospore in it. 8. That is a capsule stain and the arrow #3 is a capsule. 9. 4 is poin-ng to the bacterium. 10. The acid-fast bacterium is represented by the le_er B.

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