Why is negative staining useful for accurately determining cell size

Structure and Microscopy

Lab 4: Structure and Microscopy (100 points)

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-Each question on the lab worksheet must be answered completely, thoroughly, in complete sentences and correctly in order to be considered for full credit
-If the question asks you to do research or find a source, a reputable, credible and/or scholarly source citation must be included in order to be considered for full credit
-If a math formula is required to arrive to an answer, work must be shown otherwise, no credit will be awarded
Pre-Lab Questions
1. What determines if a bacterial cell is Gram-positive or Gram-negative? (5 points)

Amount and location of the peptidoglycan molecule in the prokaryotic cell wall determines whether a bacterial cell is Gram-positive or Gram-negative.

2. In this lab, both viruses and prions were introduced as acellular organisms. Do some research and describe one other type of acellular organism. What characteristics about this organism classify it as acellular? (5 points)

Viroids are another type of acellular organism along with viruses and prions. They are plant pathogens, which consist only of a short strand of circular RNA capable of self-replication.

3. Bacteria have many different shapes that often determine their class. Research and form a hypothesis on the evolutionary reasons for so many different bacterial morphologies. (5 points)

Each bacterial morphology may be a selectable feature to aid survival and may have affected by different physical, environmental, and biological forces to contribute to natural selection.

Reference:

Young, K. D. (2006, September). The Selective Value of Bacterial Shape. Retrieved September 30, 2018, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1594593/

4. Do a search online or look in your textbook for 1-2 antibiotics that affect Gram-positive bacteria and list them. On what part of the cell do the antibiotics usually work? List one or two antibiotics that affect Gram-negative bacteria? On what part of the cell do the antibiotics usually work? (Be sure to cite your sources in your answer.) (5 points)

5. Why do you think it is important to identify a bacterial disease in a patient before prescribing any antibiotic treatments? (Be specific.) (5 points)

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Experiment 1 Results Tables
Table 1: Experiment 1 Staining Observations (5 points)

Stain used:

Crystal Violet

Observations:

Purple rod-shape bacteria with white background were observed

Experiment 1 Post-Lab Questions
1. How does crystal violet enhance the visualization of microbial features? (5 points)

Crystal violet enhances the contrast between the microorganism itself and the slide, making the bacteria appear as purple.

2. What are some of the limitations of simple staining? (5 points)

3. Give an example of a situation in a lab or medical setting in which simple staining would be utilized. (5 points)

Simple staining is used to obtain basic information about morphology of one type of microorganism through clear visualization.

Experiment 2 Results Tables
Table 2: Experiment 2 Staining Observations (5 points)

Stain used:

Nigrosin

Observations:

Background is stained, bacteria shows up as clear spiral.

Experiment 2 Post-Lab Questions
1. After visualizing the stained samples either using your microscope or by looking at the sample images provided, describe what physical/visual characteristics you were able to observe after performing the negative staining vs. after performing the simple stain. (5 points)

After looking at the sample images provided, negatively stained bacteria showed up as clear straight spirals against a dark background. Bacteria that are simple stained showed up as dark purple rods-shaped with white background.

2. So far in this lab, you have used one type of simple stain and one type of negative stain, yet there are many other simple and negative dyes available. Pick one simple dye and one negative dye, and discuss how those dyes differ from the ones you used in this lab. Give a scenario in which their use would be appropriate. (5 points)

Methylene blue is another dye that can be used for negative stain.

India Ink is another type of negative stain.

Experiment 3 Results Tables
Table 3: Experiment 3 Staining Observations (5 points)

Stain used:

Crystal violet (primary stain) & Safranin (counterstain)

Observations:

Gram-positive appeared as purple and Gram-negative showed up as pink.

Experiment 3 Post-Lab Questions
1. What color are the Gram-positive bacteria after Gram staining? Gram-negative bacteria? (5 points)

Gram-positive bacteria appear as dark purple or blue due to retaining the primary dye (Crystal Violet) in the cell wall.

Gram-negative bacteria appear as red or pink due to decolorizing to accept the counterstain (Safranin).

2. What different characteristic(s) exist between the two groups that account for the different staining conditions? (5 points)

Gram-positive bacteria are stained purple, and gram-negative bacteria stain as pink. They are two distinct morphological groups of bacteria.

3. Why was the Gram iodine added to the Gram staining procedure? (5 points)

Gram iodine is added as a mordant to stabilize the crystal violet iodine complex so that the dye cannot be removed easily.

4. Why is a counterstain (safranin) added to the Gram staining procedure? (5 points)

A counterstain is used to help identify gram-negative bacteria. Gram-negative bacteria lose the crystal violet and stain red.

5. What are the advantages of performing a Gram stain vs. a simple stain for visualizing bacteria? (5 points)

Gram stain contains two or more different stains and can differentiate the species of bacteria into two main groups (gram-positive and gram-negative) by looking at the color of cells (pink or purple). Simple stain involves single stain and it is used to easily determine cell shape, size, and arrangement.

6. Using either a textbook or a reputable online resource, research some of the typical characteristics of bacteria, and discuss why it might be important for a researcher or a hospital technician to be able to differentiate between Gram-positive and Gram-negative bacteria. (5 points)

7. Did you experience any technical difficulties or atypical results during this experiment? If so, what happened, and how could you avoid these issues in the future? (5 points)