D311 Task 2: Identifying Unknown Microorganisms in Lab 9

Grace J.
November 12, 2025November 12, 2025
BSN, D311, Western Governors University

Student Name

Western Governors University

D311 Microbiology with Lab: A Fundamental Approach

Prof. Name

Date

MICROBIOLOGY WITH LAB: A FUNDAMENTAL APPROACH

Task 2: Unknown Lab Manual 9

Objective of Task 2

The purpose of Task 2 in this microbiology lab exercise is to accurately identify an unknown bacterial species using a limited set of laboratory tools and diagnostic tests, replicating real-world laboratory constraints. This task emphasizes the development of critical thinking and problem-solving abilities by encouraging students to analyze laboratory data, make informed choices about subsequent testing, and ultimately confirm the organism’s identity. It fosters analytical reasoning and decision-making skills crucial for understanding complex biological systems involved in microbial identification.

Task Instructions

Welcome to the Unknown 9 Lab! Your mission is to strategically apply a series of laboratory tests to determine the identity of an unknown bacterial species. Before beginning, ensure that you have selected the correct organism group based on the first letter of your last name. Since laboratory resources and time are typically limited, it is neither practical nor necessary to perform every test on every sample. You will use the provided decision tree to guide your diagnostic test selections, progressively narrowing down possible organisms until the final identification is made.

Your lab report should focus on the Gram stain and two additional tests selected based on the decision tree and previous results. Each test choice must be justified in your report. After identifying the organism, interpret the results of the Kirby-Bauer antibiotic sensitivity test to recommend an appropriate antibiotic treatment.

Investigation Tools

Familiarize yourself with Section 4, Lesson 2, which covers the Gram staining procedure and the decision tree used to guide your test choices.

Save your completed report as a Word document titled: D311 Task 2 Lab Report [Last Name]. Make sure to clearly indicate the unknown organism number to facilitate timely evaluation.

Lab Procedure Overview

Initial Step: Gram Stain

Begin by performing a Gram stain to classify the bacterium as either Gram-positive or Gram-negative. Examine cell morphology to determine if the bacterium is rod-shaped (bacillus) or spherical (coccus).

Question	Answer
Is the organism Gram-positive or Gram-negative?	Provide justification based on the color observed in the stain and cell wall structure (purple for Gram-positive, pink/red for Gram-negative).
What is the morphology of the organism?	Specify whether the cells are bacilli or cocci, supported by microscopic observations.

Next Test Selection

Following Gram stain results and guided by the decision tree, select the next appropriate test. Choose tests purposefully based on evidence from earlier observations. The decision tree includes diagnostic tests such as endospore staining, catalase, glucose fermentation, citrate utilization, blood agar hemolysis, capsule staining, motility, lactose fermentation, Voges-Proskauer, coagulase, and Kirby-Bauer antibiotic testing.

Detailed Tests and Interpretations

Endospore Stain

This test detects the formation of resistant spores by the bacterium. Spores will appear as distinct colored structures in the stain.

Question	Response
What colors are observed in the stain?	Describe the colors observed and their significance (e.g., green spores against pink vegetative cells).
Does the organism form spores?	Confirm presence or absence of spores based on the stain.
What is the next test chosen?	Justify the choice of the next test based on spore presence and decision tree recommendations.

Capsule Stain

Capsule staining helps identify if the bacterium produces a polysaccharide capsule, visible as a clear halo surrounding the cell.

Question	Response
Is there visible space around cells?	Indicate if a capsule is present by noting clear halos.
Does the organism form a capsule?	Confirm capsule presence with staining evidence.
What is the next test chosen?	Explain the selection based on capsule results.

Blood Agar Growth and Hemolysis

This test assesses the ability of the bacterium to grow on blood agar and its hemolytic activity, which can be classified as alpha (partial), beta (complete), or gamma (none).

Question	Response
Is there bacterial growth? What is the color?	Describe growth characteristics and colony coloration.
What type of hemolysis is observed?	Identify hemolysis type with supporting observations.
What is the next test chosen?	Provide reasoning for the next test based on hemolysis patterns.

Citrate Test

The citrate utilization test determines if the bacterium can use citrate as its sole carbon source, usually indicated by a medium color change.

Question	Response
What is the color of the medium?	State the color observed after incubation.
Are results positive or negative?	Interpret if the bacterium can metabolize citrate.
What is the next test chosen?	Justify the test selection based on citrate test outcomes.

Voges-Proskauer Test

This test identifies the presence of acetoin, a metabolic byproduct, by detecting a color change in the reagent tube.

Question	Response
What color is the reagent tube?	Record the color observed.
Are results positive or negative?	Interpret the presence or absence of acetoin.
What is the next test chosen?	Explain the test choice based on metabolic results.

Glucose Fermentation Test

This test measures the organism’s ability to ferment glucose, often accompanied by gas production.

Question	Response
What color is the tube? Is gas produced?	Describe color changes and presence of gas bubbles.
Are results positive or negative?	Interpret the fermentation result.
What is the next test chosen?	Provide rationale for next testing based on glucose fermentation results.

Lactose Fermentation Test

Similar to the glucose test, this evaluates lactose fermentation and gas production.

Question	Response
What color is the tube? Is gas produced?	Note tube color and presence of gas.
Are results positive or negative?	Conclude the organism’s lactose metabolism.
What is the next test chosen?	Support test choice based on lactose fermentation findings.

Catalase Test

Catalase activity is determined by observing bubbling when hydrogen peroxide is added, indicating the breakdown of peroxide.

Question	Response
Were bubbles observed?	Yes or no.
Are results positive or negative?	Explain catalase activity significance.
What is the next test chosen?	Justify the next step guided by catalase results.

Coagulase Test

This test identifies coagulase enzyme production, which causes plasma clotting.

Question	Response
Was the tube liquid or clumped?	Describe the physical state of the sample.
Are results positive or negative?	Interpret coagulase presence or absence.
What is the next test chosen?	Explain the decision based on coagulase test results.

Motility Test

This test determines whether bacterial growth is diffuse (motile) or confined (non-motile).

Question	Response
Was the growth diffuse or defined?	Describe the growth pattern.
Are results positive or negative?	Conclude on motility.
What is the next test chosen?	Provide reasoning for the next test based on motility.

Kirby-Bauer Diffusion Test

The Kirby-Bauer test measures the organism’s susceptibility to various antibiotics. Results classify the organism as resistant, intermediate, or susceptible, informing treatment choices.

Question	Response
What is the resistance profile to each antibiotic?	List susceptibility or resistance for each antibiotic tested.
Which antibiotics are unsuitable for treatment?	Identify antibiotics to avoid based on resistance.
Which antibiotic is optimal for treatment?	Recommend the best antibiotic supported by susceptibility results.

Summary

The identification of unknown bacteria in clinical microbiology requires a systematic, evidence-based approach. Beginning with the Gram stain to categorize bacteria, the use of a decision tree optimizes test selection, conserving resources while increasing diagnostic accuracy. Subsequent confirmatory tests narrow the possibilities, and the Kirby-Bauer antibiotic sensitivity test guides effective treatment decisions. This stepwise strategy combines practical lab techniques with critical analysis to ensure accurate microbial identification and appropriate clinical management.

References

Cappuccino, J. G., & Welsh, C. T. (2017). Microbiology: A Laboratory Manual (11th ed.). Pearson.

Madigan, M. T., Bender, K. S., Buckley, D. H., Sattley, W. M., & Stahl, D. A. (2018). Brock Biology of Microorganisms (15th ed.). Pearson.