D311 Microbiology Lab Report: Identification Tests & Results

D311 Microbiology Lab Report: Identification Tests & Results

Student Name

Western Governors University 

D311 Microbiology Lab Report: Identification Tests & Results

Prof. Name

Date

Microbiology Lab First Identification Test: Gram Stain

What did the Gram stain reveal about the bacterial specimen?

Microscopic examination of the Gram-stained bacterial specimen showed purple-colored cells arranged in clusters of spheres, identifying them as cocci. The purple coloration indicates that the bacteria are Gram-positive. This result is attributed to differences in bacterial cell wall structure. Gram-positive bacteria possess a thick peptidoglycan layer, making up about 90% of the cell wall. This thick layer retains the crystal violet–iodine complex during the decolorization stage, causing these bacteria to appear violet under the microscope. In contrast, Gram-negative bacteria have a thinner peptidoglycan layer and an outer membrane, which allows the crystal violet stain to be washed away, resulting in pink or red cells after counterstaining with safranin (WGU-CGP-OEX).

---

Second Identification Test: Catalase Test

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

The catalase test is conducted to differentiate Gram-positive cocci by detecting the presence of the enzyme catalase. Catalase breaks down hydrogen peroxide (H₂O₂), a harmful byproduct of aerobic metabolism, into water and oxygen. This enzyme protects bacteria from oxidative damage to critical cellular components such as DNA and proteins. During the test, bacterial culture is mixed with hydrogen peroxide. The immediate appearance of oxygen bubbles signals a positive test, confirming catalase production. The absence of bubbles indicates a negative result, showing the organism lacks catalase (WGU-CGP-OEX).

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

In Unknown Lab Manual No. 6, the catalase test was negative, as no bubbles formed upon exposure to hydrogen peroxide. This result suggests that the bacterium does not produce catalase and therefore cannot effectively neutralize reactive oxygen species. Consequently, the organism likely thrives in environments with low oxygen levels (anaerobic or microaerophilic conditions). This narrows the identification to catalase-negative Gram-positive cocci, typically belonging to genera such as Enterococcus or Streptococcus (WGU-CGP-OEX).

---

Third Identification Test: Blood Agar

What does the blood agar test assess in bacterial identification?

The blood agar test evaluates a bacterium’s capacity to lyse red blood cells by producing hemolysins—enzymes that break down red blood cells. The medium contains 5% sheep blood and nutrient agar, offering a rich environment to differentiate species based on their hemolytic activity. After incubation, the bacterial colonies are observed for changes in the color or transparency of the surrounding agar (WGU-CGP-OEX).

How are the hemolysis types distinguished?

Hemolysis Type	Description	Visual Indicator on Agar Plate
Alpha-hemolysis	Partial hemoglobin degradation producing biliverdin	Greenish halo around colonies
Beta-hemolysis	Complete red blood cell lysis	Clear, transparent zone surrounding colonies
Gamma-hemolysis	No hemolysis	No change in color or clearing of the medium

What were the blood agar results for the unknown specimen?

The bacterial culture from Unknown Lab No. 6 exhibited gamma-hemolysis, as there was no visible change in the agar surrounding the colonies. This indicates that the organism does not produce hemolysins and cannot lyse red blood cells. Gamma-hemolytic bacteria are generally nonpathogenic or opportunistic, which is consistent with the Enterococcus genus (WGU-CGP-OEX).

---

Organism Identification

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

Integrating the Gram stain (Gram-positive cocci), catalase test (negative), and blood agar test (gamma-hemolysis) results, and utilizing a bacterial classification decision tree, the unknown organism from Lab Manual No. 6 was identified as Enterococcus faecalis. This facultative anaerobe is part of the normal human gut flora but can become an opportunistic pathogen, causing infections such as urinary tract infections, bacteremia, and endocarditis in immunocompromised individuals.

---

Kirby-Bauer Diffusion Test

What is the purpose of the Kirby-Bauer test?

The Kirby-Bauer disk diffusion test is a standardized procedure used to evaluate the antibiotic susceptibility of bacterial isolates. It helps clinicians select effective antibiotics by determining if bacteria are resistant, intermediate, or susceptible to various drugs. The test measures the size of the inhibition zones around antibiotic disks placed on agar inoculated with bacteria. These zones correspond to bacterial growth suppression by the antibiotics (WGU-CGP-OEX).

How is the Kirby-Bauer test performed and interpreted?

Antibiotic-impregnated disks are placed on an agar plate evenly covered with the bacterial culture. As antibiotics diffuse from the disks, a concentration gradient forms, inhibiting bacterial growth in a zone around the disks. The diameter of these zones is measured in millimeters and compared to Clinical and Laboratory Standards Institute (CLSI) guidelines to classify the bacteria’s susceptibility.

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

*Note: Doxycycline was tested twice, yielding different zone sizes possibly due to variations in antibiotic concentration or diffusion.

What antibiotic is recommended for treating Enterococcus faecalis?

The Kirby-Bauer test revealed that doxycycline produced the largest zone of inhibition, indicating it is the most effective antibiotic against the Enterococcus faecalis isolate in Unknown Lab No. 6. Hence, doxycycline is recommended as the treatment, though clinical factors should always be taken into account.

D311 Microbiology Lab Report: Identification Tests & Results

---

References

Sign in or Register | WGU-CGP-OEX. (n.d.). Microbiology Lab: Identification Tests & Results. Retrieved from https://cgp-oex.wgu.edu/courses/course-v1:WGUx+D311+2021_T3/courseware/1b3491b45ff14c92b305d3388a8b883e/a18db52640c3474bb7b61399a115e5f0/?child=first