BIOL 2460 AO Lab Practical #1 Study Guide

Lab 1 Material

  • Broth-to-Agar Plate Transfer
    • Correct order of steps for transferring bacteria from broth culture to an agar plate:
    1. Label the agar plate properly.
    2. Use a sterile loop or swab to collect the broth culture.
    3. Streak the loop or swab on the agar surface using an appropriate streaking method.
    4. Incubate the plate under suitable conditions to allow colony growth.
  • Broth-to-Broth Transfer
    • Steps for transferring bacteria from one broth culture to another:
    1. Label the new broth tube.
    2. Use a sterile loop to pick up bacteria from the original broth tube.
    3. Insert the loop into the new broth tube and swirl to disperse the bacteria.
    4. Incubate the new broth tube appropriately.
  • Proper Use of Bunsen Burner
    • Necessary for sterilizing instruments and creating an aseptic environment:
    • Light the burner and adjust the flame to ensure it is safe and effective.
    • Use the flame to sterilize loops before inoculating, and keep the area around the flame free of materials.
  • BSL Requirements
    • Biological Safety Level (BSL) requirements must be followed depending on the organisms handled:
    • BSL-1: Basic teaching labs.
    • BSL-2: Moderate risk, contains specific protocols.
  • Definitions:
    • Sterile: Free from all living microorganisms, spores, and viruses.
    • Aseptic Technique: Procedures that prevent contamination of sterile surfaces and cultures.

Lab 2 Material

  • Calculation of Total Magnification
    • Formula: Total Magnification = Ocular Lens Magnification × Objective Lens Magnification.
  • Parts of a Microscope
    • Key components and their functions:
    • Ocular Lens: Eyepiece through which to view specimens.
    • Objective Lenses: Different magnifications for viewing (e.g., 10x, 40x, 100x).
    • Stage: Platform to hold the specimen slide.
    • Light Source: Illuminates specimen for better visibility.
  • Definition of "Resolution"
    • The ability to distinguish two points as separate entities; affected by:
    • Quality of lenses.
    • Wavelength of light.
  • Factors Affecting Resolution
    • Numerical Aperture (NA) of the lens.
    • Wavelength of light used; shorter wavelengths improve resolution.

Lab 3 Material

  • Fixing Bacteria to a Slide
    • Process involves heat-fixing:
    • Pass the slide through flame to kill and adhere the bacteria.
  • Gram Stain Method
    • Procedure includes:
    1. Crystal violet application.
    2. Iodine treatment.
    3. Decolorization with alcohol.
    4. Counterstaining with safranin.
    • Interpretation of Gram Stain Results:
    • Gram-positive: Retain crystal violet (appear purple).
    • Gram-negative: Lose color and take up safranin (appear pink).
  • Acid Fast Stain Method
    • Use to identify acid-fast bacteria such as Mycobacterium:
    1. Carbol fuchsin application.
    2. Decolorization with acid-alcohol.
    3. Methylene blue counterstaining.
    • Interpretation of Acid Fast Stain Results:
    • Acid-fast organisms appear red; non-acid-fast appear blue.
  • Endospore Stain Method
    • Used for identifying endospore-forming bacteria:
    1. Malachite green application (with heat).
    2. Decolorization with water.
    3. Counterstain with safranin.
    • Interpretation of Endospore Stain Results:
    • Spores appear green; vegetative cells appear pink.

Lab 4 Material

  • Two Streak Plate Method
    • Technique for isolating pure cultures from a mixed culture:
    • Sterilize the loop between streaks, transferring only a small amount from one quadrant to the next.
  • Spread Plate Method
    • Involves spreading a diluted sample over the surface of an agar plate:
    • Used to count Colony Forming Units (CFUs) and isolate individual colonies.
  • Colony Forming Unit (CFU)
    • Definition:
    • A unit used to estimate the number of viable bacteria or fungal cells in a sample. One CFU is assumed to arise from a single organism.