BIO 139 Lab Exam II Study Notes

Lab Exam Overview

Subject: BIO 139 Lab

Instructor: Bendorf

Date: April 30, 2025

Coverage: Weeks 8 - 14 of lab material, which includes in-depth exploration of microbial techniques and their applications in real-world scenarios.

Weight: 30 points, contributing to your final grade in the course based on performance across various formats.

Format: Multiple choice, fill in the blank, and short essay questions similar to quizzes, including questions that require critical thinking and application of lab knowledge.

Calculations: CFU/PFU from dilutions for analyzing microbial concentrations; students are allowed to use scientific calculators, but smart devices are prohibited due to potential academic dishonesty.

Water Quality

Calculating Original Concentration:

  • Based on colony counts from diluted agar plate samples, which assess the level of microbial presence in water samples.

  • Proper units to express concentration include CFU/mL, CFU/sample, CFU/L, ensuring clarity in quantitative microbiology.

  • Conversions between units such as L to mL to μL are crucial for accurate calculations in diverse laboratory environments.

Staining and Gram Reaction Confirmation

KOH Test:

  • Distinguishes between Gram positive and Gram negative cells by analyzing the cell wall structure, which affects their response to antibiotics.

Gram Staining Procedure:

  • Recall the steps (crystal violet stain, iodine treatment, decolorization, and counterstaining), and troubleshoot methods in case of inconsistencies in expected results.

Flagella Stain:

  • Principle: Visualizes the presence and arrangement of flagella structures on bacterial cells.

  • Results:

    • Positive: Flagella visible under a light microscope, indicating motility capabilities.

    • Negative: Flagella not visible, suggesting either the absence of motility or issues with the staining process.

  • Explanation: Discuss biological and technical reasons for variations in results, contributing to understanding bacterial behavior.

Selective and Differential Media

Purpose: Understand when and why to use specific media types in isolating and identifying distinct microbial species.

Examples of Media & Agents:

  • MacConkey agar: Selects for Gram-negative bacteria and differentiates lactose fermenters based on color change.

  • Nutrient agar: Provides essential nutrients for general bacterial growth.

  • Casein agar: Used to detect proteolytic activity in bacteria.

  • Rhodamine B agar: Used to visualize bacteria that can metabolize certain substrates, commonly used in water quality testing.

Oxygen Requirements

Microbial Types:

  • Obligate aerobes: Require oxygen for growth due to aerobic respiration.

  • Aerotolerant microbes: Can survive in oxygen but do not use it for growth.

  • Facultative anaerobes: Prefer oxygen but can grow in its absence by fermentation or anaerobic respiration.

  • Obligate anaerobes: Cannot survive in the presence of oxygen; utilize fermentation or other anaerobic processes for energy.

Environmental O2 Differences:

  • The atmosphere, which has approximately 21% oxygen, provides a baseline for aerobic conditions.

  • Candle jar: A method to create a low-oxygen environment for incubating certain bacteria.

  • Anaerobe jar: Specifically designed to cultivate anaerobic bacteria, removing oxygen to allow obligate anaerobes to thrive.

Phenotype Tests

Test Preparation:

  • Know principles, positive/negative results, and their meanings in assessing microbial characteristics.

  • For negatives, consider possible biological and technical explanations that may impact results.

List of Tests:

  • Motility: Assesses bacterial motility in semi-solid media.

  • Crystal violet biofilm assay: Evaluates biofilm formation on surfaces.

  • Phage assay: Determines the presence and activity of bacteriophages.

  • Calculate PFU concentrations from dilutions to measure viral populations.

Biochemical Tests

Understand for each test:

  1. Reactants: The substances that undergo a chemical change during the test.

  2. Products: The resulting substances after the reaction.

  3. Detection methods: Techniques such as Durham tubes for gas detection and pH changes for metabolic activity.

  4. Interpretation of results: Understanding what positive and negative results signify in microbial metabolism and identification.

Specific Tests:

  • BCP Sugars Test: Indicates carbohydrate fermentation and acid production.

  • Nitrate Reduction/Denitrification Test: Assesses the ability of microbes to reduce nitrate to nitrite or nitrogen gas.

  • Catalase Test: Principles and expected results indicating the breakdown of hydrogen peroxide in cultures.

  • Oxidase Test: Principles and expected results to determine cytochrome c oxidase presence in bacteria.

  • Gelatinase Test: Detects the ability of bacteria to hydrolyze gelatin, indicating potential pathogenicity.

Troubleshooting Experiments

Analyze unexpected results or absence of results thoroughly; consider pitfalls in the experimental process.
Identify potential reasons for discrepancies such as contamination or procedural errors.
Suggest adjustments to improve experimental outcomes and support learning from mistakes to refine practical skills for future experiments.

The KOH test involves the following steps to distinguish between Gram positive and Gram negative cells based on their cell wall structure:

  1. Preparation of the Sample:

    • Take a small amount of the bacterial colony to be tested.

  2. Mix with KOH Solution:

    • Place the colony into a drop of potassium hydroxide (KOH) solution on a clean glass slide.

  3. Observation:

    • Mix the sample with the KOH using a stick or stirrer and observe the consistency of the mixture.

  4. Results Determination:

    • If the mixture becomes viscous and stringy, the test is positive for Gram negative bacteria.

    • If the mixture remains watery without string formation, the test indicates Gram positive bacteria.

This test informs about the cell wall's structural properties, influencing the effectiveness of certain antibiotics.

Recall the steps involved in the Gram staining procedure, which include:

  1. Crystal Violet Staining:

    • Apply crystal violet stain to the bacterial smear, allowing it to penetrate the cell walls.

  2. Iodine Treatment:

    • Apply iodine, forming complexes with the crystal violet, which helps in the adherence of the dye to the cells.

  3. Decolorization:

    • Use alcohol or acetone to decolorize the cells, differentiating between Gram positive and Gram negative bacteria based on their cell wall structure.

  4. Counterstaining:

    • Apply a counterstain (such as safranin) to visualize the decolorized Gram negative cells.

Troubleshoot methods in case of inconsistencies in expected results. Potential issues may include over-decolorization or under-staining, affecting the determination of bacterial Gram status.