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Bacteria Lab Worksheet

This document contains a student's lab worksheet analyzing Gram staining and antibiotic sensitivity testing of two bacteria - E. coli and S. aureus. Key findings include: E. coli is Gram-negative and rod-shaped while S. aureus is Gram-positive and pentagon/hexagon shaped. E. coli absorbed the counterstain safranin because it was originally colorless. Gram-positive bacteria have a thicker peptidoglycan cell wall layer compared to Gram-negatives, which have an additional outer membrane containing lipopolysaccharides. The most effective antibiotic against S. aureus was Tetracycline based on the size of the zones of inhibition in the Kirby-Bauer test.

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harrison
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1K views

Bacteria Lab Worksheet

This document contains a student's lab worksheet analyzing Gram staining and antibiotic sensitivity testing of two bacteria - E. coli and S. aureus. Key findings include: E. coli is Gram-negative and rod-shaped while S. aureus is Gram-positive and pentagon/hexagon shaped. E. coli absorbed the counterstain safranin because it was originally colorless. Gram-positive bacteria have a thicker peptidoglycan cell wall layer compared to Gram-negatives, which have an additional outer membrane containing lipopolysaccharides. The most effective antibiotic against S. aureus was Tetracycline based on the size of the zones of inhibition in the Kirby-Bauer test.

Uploaded by

harrison
Copyright
© © All Rights Reserved
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Download as DOCX, PDF, TXT or read online on Scribd
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Name: Harrison Dempsey

Date: 1/13/21

Bacteria Lab Worksheet


Part One: Gram Staining Analysis

1. Observe the micrograph above containing two different bacteria on one slide. The two bacteria are
Escherichia coli (E. Coli) and Staphylococcus Aureus (S. Aureus). After completing the Gram staining
process, the E. Coli is determined to be Gram-negative while the S. Aureus is Gram-positive. Answer the
following questions:

a. What is the shape of the S. Aureus? What is the shape of the E. Coli? (3 pts)
S. Aureus is pentagon/hexagon shaped and the E. Coli is rod shaped.

b. What color is the E.Coli? What color is the S. Aureus? (3 pts)


E. Coli- Pink

S. Aureus- Purple
c. Which bacteria absorbed the counterstain (safranin)? Why? Explain in at least 3 well-written
sentences. (6 pts)
E. Coli absorbed the counterstain. This is because the bacteria originally are the same color.
Therefore, it must be stained a different color so you can compare which one is which.

d. Which bacteria is more resistant to antibiotics? Why? Explain in at least 3 well-written sentences.
(6 pts)
E. Coli is more resistant to antibiotics. This is because of the zones of inhibition. These zones
mean the antibiotic was effective against the bacteria.

2. Gram-Positive Bacteria Analysis

a. What carbohydrate comprises 90% of the cell wall? (3 pts)


Peptidoglycan

3. Gram-Negative Bacteria Analysis

a. In the extra outer membrane, lipopolysaccharides are considered to be endotoxins. What does this
mean? (4 pts)
“An endotoxin is a harmful substance which releases toxins from inside a bacterial cell as the cell
breaks down. It is this outer membrane that prevents certain medicines and antibiotics from
penetrating the cell wall(Today’s Lab)” This means if anything tried to get into the cell, this
endotoxin would fight it off.

(continue below)
Part Two: Antibiotic Sensitivity Analysis

1. Observe the Petri dish above containing the bacteria Staphylococcus aureus (S. aureus). Answer the
following questions based on the results from the Kirby-Bauer test.

a. Rank the antibiotics from 1-5 starting with the most effective (1) to least effective (5) against the S.
aureus in the chart below: (10 pts)

1. Tetracycline
2. Streptomycin
3. Neomycin
4. Lincomycin
5. Nystatin

(continue below)
b. How can you tell how effective an antibiotic is against a bacteria just by looking at the Petri dish?
Explain in 3-5 sentences. (10 pts)
I can tell the difference by the zones of inhibition. These zones are places where the bacteria died
or didn’t grow at all. The more effective the antibiotic is, the bigger the zone of inhibition will be.
In the picture above, Tetracycline was the most effective and Nystatin was least effective.

Conclusions: Gram Staining


1. During the Gram staining process, what can be said about a bacteria if it does not absorb the
counterstain (safranin)? Would it be Gram-positive or Gram-negative? How can you tell? (5 pts)
If a bacteria does not absorb the counterstain, it would be gram positive. This is because gram-
positive cells retain the purple dye-mordant complex because of their thick layer of peptidoglycan.

2. What is unique about the composition of the cell wall of Gram-negative bacteria? (5 pts)
In the Gram-negative Bacteria the cell wall is composed of a single layer of peptidoglycan surrounded by
a structure called the outer membrane. The outer membrane of Gram-negative bacteria contains
a unique component, lipopolysaccharide, in addition to proteins and phospholipids.

3. What is known as the principal stain? (5 pts)


Principle stain is known as “Crystal Violet”

“Crystal violet is used to make bacteria visible and easy to identify under a microscope(Today’s Lab) ”

4. Why are Gram-positive bacteria unaffected by the counterstain (safranin)? Explain in 3-5 sentences.
(10 pts)
In step 7, The gram positive bacteria is purple while the gram negative bacteria is colorless. When
the safranin is added to the slide, the gram positive bacteria is not effected. Gram positive bacteria
is not effected because it already has a purple color, and the gram negative has no color. Therefore,
the counterstain does not add color to the gram positive bacteria.

(continue below)
5. What happens when heat is applied to a slide containing bacteria? What is the proper term for this
process? (5 pts)
When heat is applied to the slide, it kills any bacteria that could compromise the results. This
process is known as “Heat Fixation.”

6. In two well-constructed paragraphs, compare and contrast the cell walls of Gram-positive and Gram-
negative bacteria. (15 pts)
Gram positive and gram negative bacteria have a cell wall made up of peptidoglycan. Both cells
contain a periplasmic space as well as a plasma membrane. Both of these bacteria cells also have an
outer membrane that helps protect the cell.

Although Gram negative bacteria also has a wall made up of peptidoglycan, but there is much less of
it. This means the wall is a lot thinner than the wall in the gram positive bacteria. The gram positive
bacteria wall is much thicker. Gram negative cells have a double layered cell wall as gram negative
cells only have one layer.

Conclusions: Antibiotic Sensitivity


1. What is the importance of studying antibiotic sensitivity in bacteria? (5 pts)
It is important to study antibiotic sensitivity in bacteria because Antibiotics are one of mankind's
most important discoveries. They allow us to survive serious bacterial infections. When bacteria
become resistant to an antibiotic, it means that the antibiotic can no longer kill that bacteria. If an
antibiotic can not kill the bacteria of need, humans would never recover from sickness.

2. What are the two names mentioned for the test which examines antibiotic sensitivity? (5 pts)
Gram Staining Method and Kirby Bauer Test

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