D311 Task 2: Identifying Unknown Microorganisms in Lab 9

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

The objective of Task 2 in this microbiology lab is to identify an unknown bacterial organism using a minimal number of laboratory instruments and tests, simulating real-world constraints. This process emphasizes problem-solving skills by encouraging learners to analyze provided lab evidence, make informed decisions about subsequent tests, and ultimately confirm the organism’s identity. The exercise aims to develop critical thinking, analytical reasoning, and decision-making capabilities, which are essential for connecting complex biological systems during microbial identification.

Task Instructions

Welcome to the Unknown 9 Lab! Your task is to apply various laboratory tests strategically to identify an unknown bacterial species. Prior to starting, ensure you have selected the correct organism group corresponding to the initial letter of your last name. Given the typical limitations of lab resources and time, not every test should be performed on every sample. Use the provided decision tree to guide your selection of diagnostic tests, narrowing possibilities until a final identification is reached.

In your lab report, focus your discussion on the Gram stain and two other tests chosen based on your results and decision tree guidance. Justify your choice for each test within the report. Upon identification of the organism, you will interpret the results of the Kirby-Bauer antibiotic sensitivity test to recommend an appropriate treatment option.

Investigation Tools

Review Section 4, Lesson 2 for background on Gram staining and to familiarize yourself with the decision tree that will guide test selection.
Save your lab report as a Word document titled: D311 Task 2 Lab Report [Last Name].
Clearly indicate the unknown organism number you are identifying to avoid delays in evaluation.

Lab Procedure Overview

Initial Step: Gram Stain
Start identification by performing a Gram stain to classify the bacterium as Gram-positive or Gram-negative. Observe the morphology to determine if the cells are bacillus (rod-shaped) or coccus (spherical).

Guiding Questions:

Question	Answer
Is the organism Gram-positive or Gram-negative?	Provide justification based on staining color and cellular structure.
What is the morphology of the organism?	State whether it is bacillus or coccus and support with visual evidence.

Next Test Selection
Based on Gram stain results and the decision tree, choose the next test. This choice should be purposeful, considering the evidence from prior tests. The decision tree includes options 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 reveals whether the organism produces resistant spores. The presence of spores is indicated by distinct coloration in the staining process.

Guiding Questions	Responses
What colors are observed in the stain?	Describe the colors and their significance.
Does the organism form spores?	Confirm based on the presence of spores in the sample.
What is the next test chosen?	Explain based on decision tree and spore results.

Capsule Stain

Capsule staining identifies the presence of a polysaccharide capsule surrounding the bacterium, visible as a clear halo around the cell.

Guiding Questions	Responses
Is there visible space around cells?	Note the presence or absence of capsule.
Does the organism form a capsule?	Confirm with evidence from the stain.
What is the next test chosen?	Justify the selection based on capsule presence.

Blood Agar Growth and Hemolysis

This test assesses bacterial growth on blood agar and hemolytic activity, classifying hemolysis into alpha (partial), beta (complete), or gamma (none).

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

Citrate Test

This assay determines if the bacterium can utilize citrate as a carbon source, typically indicated by a color change in the medium.

Guiding Questions	Responses
What is the color of the medium?	State color observed.
Are results positive or negative?	Interpret the result relative to organism metabolism.
What is the next test chosen?	Explain selection considering citrate test outcome.

Voges-Proskauer Test

This test detects acetoin production, a metabolic byproduct, with positive results indicated by a color change in the reagent tube.

Guiding Questions	Responses
What color is the reagent tube?	Record color observed.
Are results positive or negative?	State the interpretation.
What is the next test chosen?	Justify based on the metabolic implications.

Glucose Fermentation Test

This test evaluates the organism’s ability to ferment glucose, with gas production often noted as a secondary indicator.

Guiding Questions	Responses
What color is the tube? Is gas produced?	Describe color and presence of gas bubbles.
Are results positive or negative?	Interpret the outcome for organism identification.
What is the next test chosen?	Explain rationale according to decision tree.

Lactose Fermentation Test

Similar to glucose fermentation, this test assesses lactose utilization and gas production.

Guiding Questions	Responses
What color is the tube? Is gas produced?	Note tube color and gas presence.
Are results positive or negative?	Conclude the metabolic ability.
What is the next test chosen?	Support choice based on findings.

Catalase Test

Catalase activity is demonstrated by bubbling when hydrogen peroxide is applied, indicating breakdown of peroxide.

Guiding Questions	Responses
Were bubbles observed?	Yes or no.
Are results positive or negative?	Explain meaning of catalase activity.
What is the next test chosen?	Justify selection based on catalase result.

Coagulase Test

This test detects the enzyme coagulase, which causes clot formation in plasma.

Guiding Questions	Responses
Was the tube liquid or clumped?	Describe physical state.
Are results positive or negative?	Interpret coagulation result.
What is the next test chosen?	Explain next step guided by results.

Motility Test

Motility testing observes whether bacterial growth is diffuse (motile) or confined (non-motile).

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

Kirby-Bauer Diffusion Test

This antibiotic sensitivity test determines the susceptibility of the organism to specific antibiotics. Results categorize the organism as resistant, intermediate, or susceptible, guiding therapeutic decisions.

Guiding Questions	Responses
What is the resistance profile to each antibiotic?	List susceptibility results.
Which antibiotics are unsuitable for treatment?	Identify based on resistance.
Which antibiotic is optimal for treatment?	Provide evidence supporting this choice.

Summary

In a clinical microbiology context, the identification of unknown bacterial organisms requires a strategic, stepwise approach using key laboratory tests selected through evidence-based decision-making. Starting with the Gram stain, the use of a decision tree helps streamline testing, conserving resources while enhancing diagnostic accuracy. The Kirby-Bauer test further assists in selecting appropriate antimicrobial therapy, completing the diagnostic and treatment planning cycle.

References

Western Governors University. (n.d.). Microbiology with Lab: A Fundamental Approach. Task 2 Unknown Lab Manual.
Madigan, M. T., Bender, K. S., Buckley, D. H., Sattley, W. M., & Stahl, D. A. (2018). Brock Biology of Microorganisms (15th ed.). Pearson.