Lab Exam


1. Introduction to Tiny Earth

  • Goals of Tiny Earth: Understand the project’s mission to address the global antibiotic crisis by identifying soil bacteria that produce antibiotics.

  • Soil Crisis: The degradation of soil health due to various factors, impacting its ability to sustain life.

  • Antibiotic Crisis: The growing concern of antibiotic resistance and the search for new antibiotics.

2. Lab Notebook Protocol

  • Proper Lab Notebook Keeping: Always date entries, write down protocols, and include all observations.

  • Why it’s Important: Keeps track of procedures, results, and ensures reproducibility and accountability.

3. Aseptic Technique

  • Definition: Preventing contamination by microorganisms.

  • Importance: Essential for accurate, reliable experiments and maintaining safety.

  • Tips: Flame sterilize tools, wear gloves, and work near a flame to create an upward airflow.

4. Soil Sampling Strategy & Protocol

  • Soil Sampling: Know how to collect representative soil samples and understand the purpose behind the sampling process.

  • Methods: Review the soil sampling protocols in your lab manual, such as sterilization techniques for sampling tools.

5. Serial Dilutions & Cell Density Calculations (CFU/g)

  • Serial Dilutions: How to dilute a sample step-by-step and calculate the number of bacteria in the original sample.

  • CFU/g Calculation: Practice how to calculate Colony Forming Units per gram of soil (CFU/g) from dilution series data.

6. Types of Media Used in Lab

  • Why Different Media Types are Useful: Some media promote growth of certain bacteria while inhibiting others. Be familiar with selective and differential media.

  • Media Types in Lab: Know the types used for soil bacteria isolation and antibiotic screening.

7. Colony Morphology Terminology

  • Terms to Describe Colonies: Review descriptive terms like shape, size, color, texture, and elevation for soil isolate colonies.

8. Protocols for Making Master Plates & Streak Plates

  • Purpose of Techniques: Master plates help preserve bacterial isolates, while streak plates are used to isolate individual colonies.

  • How to Make & Interpret: Review how to streak for isolation and how to interpret colony growth and morphology.

9. ESKAPE Pathogens & Safe Relatives

  • What are ESKAPE Pathogens: These are a group of antibiotic-resistant bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species).

  • Gram Stain: Know whether these pathogens are Gram-positive or Gram-negative, and why they are of interest for antibiotic research.

10. Screening Soil Bacteria for Antibiotic Production

  • Protocol Overview: Know the steps for screening bacteria for antibiotic production using the ESKAPE pathogens.

  • Results Interpretation: Understand how to determine if a bacterium is an antibiotic producer based on inhibition zones around colonies.

11. Gram Staining

  • What is Gram Staining: A method to differentiate bacteria based on cell wall structure.

  • Steps in Gram Staining: Crystal violet, iodine, alcohol wash, and safranin. Understand each step.

  • Result Interpretation: Purple = Gram-positive, Pink = Gram-negative. Be able to identify organisms based on the result.

12. Polymerase Chain Reaction (PCR)

  • How PCR Works: Amplifies specific DNA sequences.

  • Reagents Needed: DNA, primers, polymerase, dNTPs, buffer.

  • Why it’s Useful: Allows researchers to make many copies of a specific DNA region for analysis.

  • Gene Amplified in Class: Review the specific gene you amplified during the lab.

13. Agarose Gel Electrophoresis

  • How It Works: Separates DNA fragments based on size using an electric field.

  • Interpreting Results: Be able to look at a gel and identify whether a PCR reaction was successful (i.e., were the bands the expected size?).

  • Estimating Size: Learn how to compare band positions to a DNA ladder to estimate PCR product size.

14. Biochemical Tests

Be familiar with the 7 tests we used in lab, as well as any other tests presented by your classmates. Understand the following for each:

  • What is Tested For: The biological or chemical characteristic being tested.

  • What a Positive Result Looks Like: The color change, growth pattern, or other indicator of a positive result.

  • What a Negative Result Looks Like: No change or a different color.

  • How to Perform the Test: The basic steps of performing each test.

  • Selective and Differential Properties: What makes the media selective or differential.

Tests to Focus On:

  • Agar Deep Stabs: Used to test for anaerobic growth.

  • Triple Sugar Iron Agar Slants: Tests for the ability to ferment sugars and produce hydrogen sulfide.

  • Blood Plates: Tests for hemolysis (destruction of red blood cells).

  • Catalase Test: Tests for the presence of the enzyme catalase, which breaks down hydrogen peroxide.

  • MacConkey Agar: Selects for Gram-negative bacteria and differentiates lactose fermenters.

  • Starch Plates: Tests for the ability to hydrolyze starch using the enzyme amylase.

  • Motility Agar: Tests for bacterial motility, indicated by a diffuse growth pattern.