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Western Governors University
D311 Microbiology Lab Report: Identification Tests & Results
Prof. Name
Date
What did the Gram stain reveal about the bacterial specimen?
Microscopic examination of the Gram-stained bacterial specimen showed that the cells were purple and organized in spherical clusters. These features indicate that the bacteria are cocci in shape. The purple coloration retained after the staining process confirms that the bacteria are Gram-positive.
This outcome stems from differences in bacterial cell wall structures. Gram-positive bacteria have a thick peptidoglycan layer, making up around 90% of the cell wall, which traps the crystal violet–iodine complex during decolorization. As a result, Gram-positive bacteria appear blue or violet under the microscope. In contrast, Gram-negative bacteria, with a thinner peptidoglycan layer and an outer membrane, do not retain the crystal violet stain and instead take up the counterstain (safranin), appearing pink or red (WGU-CGP-OEX).
Why is the catalase test performed, and what does it indicate about the bacteria?
The catalase test is conducted after the Gram stain to differentiate among Gram-positive cocci. This biochemical assay detects the presence of catalase, an enzyme that breaks down hydrogen peroxide (H₂O₂) into water and oxygen. Hydrogen peroxide is harmful to cells because it damages DNA, proteins, and lipids.
In the test, a bacterial sample is exposed to hydrogen peroxide. If catalase is present, oxygen bubbles form rapidly, indicating a positive result. If no bubbles form, the test is negative, meaning the bacterium does not produce 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 appeared when hydrogen peroxide was applied. This indicates the bacterium lacks catalase and is less capable of neutralizing reactive oxygen species, making it vulnerable to oxidative stress. Such bacteria typically thrive in anaerobic or low-oxygen environments. This result narrows the identification to catalase-negative Gram-positive cocci, commonly found in genera like Enterococcus or Streptococcus (WGU-CGP-OEX).
What does the blood agar test assess in bacterial identification?
The blood agar test determines whether bacteria produce hemolysins, enzymes that lyse red blood cells (RBCs). The medium contains 5% sheep blood and nutrients, allowing for differentiation based on hemolytic activity. After incubation, the presence or absence of hemolysis is observed by changes in the agar surrounding bacterial colonies (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 lysis of RBCs | Clear, transparent zones around colonies |
| Gamma-hemolysis | No RBC lysis | No color change or clearing in medium |
What were the blood agar results for the unknown specimen?
The bacterial culture from Unknown Lab No. 6 showed gamma-hemolysis, as no changes in the agar surrounding colonies were observed. This indicates that the bacterium does not produce hemolysins and cannot lyse red blood cells. Gamma-hemolytic bacteria are generally nonpathogenic or opportunistic pathogens, consistent with the genus Enterococcus (WGU-CGP-OEX).
Based on the tests performed, what is the identity of the bacterial organism?
By integrating the results from the Gram stain (Gram-positive cocci), catalase test (negative), and blood agar test (gamma-hemolysis), and using the Decision Tree classification method, the unknown bacterium in Lab Manual No. 6 was identified as Enterococcus faecalis.
Enterococcus faecalis is a facultative anaerobe commonly present in the human gut microbiota. While often harmless in healthy hosts, it can act as an opportunistic pathogen, causing infections such as urinary tract infections, bacteremia, and endocarditis, particularly in immunocompromised individuals.
What is the purpose of the Kirby-Bauer test?
The Kirby-Bauer disk diffusion test evaluates the antibiotic susceptibility of bacterial isolates. This test helps clinicians choose effective antibiotics by measuring bacterial resistance or sensitivity to different drugs. The diameter of growth inhibition zones around antibiotic disks is compared with standardized charts to classify bacterial responses as susceptible, intermediate, or resistant (WGU-CGP-OEX).
How is the Kirby-Bauer test performed and interpreted?
In this test, antibiotic disks are placed on agar plates evenly inoculated with bacteria. Antibiotics diffuse into the medium, creating concentration gradients. Where antibiotic concentration inhibits bacterial growth, clear zones (zones of inhibition) appear. These zones are measured in millimeters and interpreted based on Clinical and Laboratory Standards Institute (CLSI) guidelines to determine resistance patterns.
| Antibiotic | Zone of Inhibition Result | Interpretation |
|---|---|---|
| Amoxicillin | Very small or absent zone | Resistant |
| Doxycycline | Intermediate-sized zone | Intermediate sensitivity |
| Doxycycline* | Large inhibition zone | Susceptible |
Note: Doxycycline was tested twice, showing different inhibition zones, possibly due to variation in concentration, diffusion, or procedural factors.
What antibiotic is recommended for treating Enterococcus faecalis?
Based on the Kirby-Bauer results, doxycycline demonstrated the largest zone of inhibition, indicating it is the most effective antibiotic against the Enterococcus faecalis isolate from Unknown Lab No. 6. Therefore, doxycycline is recommended as the treatment of choice, but clinical judgment and patient-specific considerations must guide final decisions.
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