D311 Microbiology Lab Report: Identification Tests & Results

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Western Governors University

D313 Anatomy and Physiology II with Lab

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Date

Microbiology Lab First Identification Test: Gram Stain

What did the Gram stain reveal about the bacterial specimen?

Upon microscopic examination of the Gram-stained specimen, the bacterial cells appeared purple and arranged in spherical clusters. These morphological characteristics identify the bacteria as cocci. The purple coloration indicates that the bacteria are Gram-positive. This staining outcome results from the structural differences in their cell walls. Specifically, Gram-positive bacteria have a thick peptidoglycan layer, which accounts for roughly 90% of their cell wall composition. This thick layer retains the crystal violet stain, causing the bacteria to appear blue or purple under the microscope (WGU-CGP-OEX).

Second Identification Test: Catalase Test

Why is the catalase test performed, and what does it indicate about the bacteria?

The next diagnostic step performed on the Gram-positive cocci was the catalase test. This test is essential to determine if the bacteria produce the enzyme catalase. Catalase breaks down hydrogen peroxide, a toxic byproduct that can damage bacterial cells. During the test, bacterial cells are mixed with hydrogen peroxide on a slide or test tube. A positive result is indicated by rapid formation of oxygen bubbles, while the absence or scarcity of bubbles denotes a negative test (WGU-CGP-OEX).

What were the catalase test results for the unknown sample, and what do they imply?

In the Unknown Lab Manual number six, the catalase test was negative, as no bubbles appeared after the addition of hydrogen peroxide. This suggests that the bacteria lack the catalase gene and are unable to neutralize hydrogen peroxide. Consequently, such bacteria are vulnerable to oxidative damage affecting vital components like lipids, DNA, and proteins. These catalase-negative bacteria typically thrive in oxygen-free environments, classifying them as anaerobic species (WGU-CGP-OEX).

Third Identification Test: Blood Agar

What does the blood agar test assess in bacterial identification?

The third test involved using blood agar to evaluate the bacterial organism’s ability to produce hemolysins—enzymes that lyse red blood cells. Blood agar contains 5% sheep blood and nutrients that support bacterial growth. After incubating the bacteria overnight, the medium is examined for hemolytic activity, classified as alpha, beta, or gamma hemolysis (WGU-CGP-OEX).

How are the hemolysis types distinguished?

Hemolysis Type	Description	Visual Indicator on Agar Plate
Alpha-hemolysis	Partial hemoglobin breakdown with biliverdin production	Greenish discoloration around colonies
Beta-hemolysis	Complete lysis of red blood cells	Clear, transparent zones around colonies
Gamma-hemolysis	No hemolytic activity	No change in the agar around colonies

What were the blood agar results for the unknown specimen?

The bacterial sample from Unknown Lab Manual number six exhibited gamma-hemolysis, showing no hemolytic activity. This indicates the organism does not produce hemolysins and cannot degrade red blood cells (WGU-CGP-OEX).

Organism Identification

Based on the tests performed, what is the identity of the bacterial organism?

Using the results of the Gram stain, catalase test, and blood agar test, combined with the Decision Tree method, the unknown bacterial specimen was identified as Enterococcus faecalis.

Kirby-Bauer Diffusion Test

What is the purpose of the Kirby-Bauer test?

The Kirby-Bauer test is a disk diffusion method used to determine the sensitivity or resistance of bacteria to different antibiotics. This test aids in selecting effective treatments by assessing how well an antibiotic inhibits bacterial growth (WGU-CGP-OEX).

How is the Kirby-Bauer test performed and interpreted?

Antibiotic-impregnated disks are placed on a bacterial culture plate. As the antibiotic diffuses outward, it creates a concentration gradient. A “zone of inhibition” forms where bacterial growth is prevented. The diameter of this zone indicates susceptibility: larger zones mean higher sensitivity, while smaller or no zones indicate resistance (WGU-CGP-OEX).

Antibiotic	Zone of Inhibition Result	Interpretation
Amoxicillin	Very small or absent zone	Resistant
Doxycycline	Intermediate-sized zone	Intermediate sensitivity
Doxycycline*	Large zone	Susceptible

*Note: Doxycycline was tested twice with varying results, possibly reflecting concentration differences or testing variations.

What antibiotic is recommended for treating Enterococcus faecalis?

Based on the Kirby-Bauer test outcomes, doxycycline is the most effective antibiotic for treating Enterococcus faecalis from the Unknown Lab Manual number six.

References

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