CH

microm 302: week 1 (Laboratory Techniques and Case Study Notes)

Ubiquity of Microorganisms

  • Microorganisms (bacteria, protozoa, fungi, etc.) are found in every environment.

Broth Culture

  • Definition: A liquid medium designed for bacterial growth.

  • Key Points:

    • Provides necessary nutrients for bacteria to multiply.

    • Agar alone is not sufficient to culture bacteria; it requires added nutrients.

Agar Types

  • TSY Agar (Tryptic Soy Yeast):

    • Nutrient-rich solid medium used for culturing organisms.

    • Agar provides a gel base; nutrients must be included for growth.

Wet Mounts

  • Purpose: To observe live motile organisms through a microscope.

  • Organisms typically observed: Bacteria, protozoa, and possibly algae.

    • Protozoa: Eukaryotic organisms (possess a nucleus and organelles).

    • Bacteria: Prokaryotic organisms (lack nucleus and organelles).

  • Sterilization: Boiling slides and coverslips post-use serves to sterilize and kill organisms before disposal.

  • Dark Field Stop: Enhances contrast, making organisms appear bright against a dark background.

Microscopy

  • Light Microscope: Utilizes visible light for observation.

    • Maximum Magnification: Approximately 1000x.

    • Total Magnification Formula: ext{Total Magnification} = ext{Objective lens} imes ext{Ocular lens}

    • Oil Immersion: Not used at 40x magnification to prevent lens damage.

Aseptic Technique

  • Main Purposes:

    1. Avoid contamination of cultures.

    2. Prevent contamination of the environment and the scientist.

  • Loop Transfer: Method used for transferring cultures.

  • Quantitative Sample: Measures specific volume for estimating cell concentration.

Making a Slide Smear

  • Heat Fixing: Kills bacteria and adheres them to the slide; no boiling required, can wash with soap and water.

    • Liquid Culture: Apply directly to the slide.

    • Solid Culture: Dilute with water first for less dense sample.

Simple Stain

  • Purpose: Increases contrast for visibility of cell shape and arrangement.

  • Contrast vs. Resolution:

    • Contrast: Difference between sample and background.

    • Resolution: Ability to discern details between two close points.

  • Basic Dyes: Positively charged dyes that bind to negatively charged bacterial cell walls.

    • Common stains: Methylene blue, crystal violet, safranin, malachite green.

  • Simple Stain Steps:

    1. Dilute solid bacterial sample in water.

    2. Heat fix the slide.

    3. Add stain (e.g., methylene blue for Spirillum).

    4. Rinse with tap water and dry.

    5. View under the microscope starting with low objective; switch to oil immersion for 100x view.

Case Study: Patient Background

  • Patient: Sarah, 25 years old, with UTI symptoms (frequent urination, burning sensation, abdominal pain).

  • Diagnosis Process:

    1. Urine sample is collected for culture (bladder should typically be sterile; contrast with intestine microbiota).

    2. Urine should not be placed directly under a microscope; must be cultured first.

    3. Urine inoculated into broth, followed by agar growth for colony isolation.

Use and Application of a Simple Stain in UTI

  • Application: Perform a simple stain (using crystal violet) to determine presence of bacteria in urine.

  • Limitations:

    • Simple stains are not diagnostic for gram properties but confirm bacterial presence in sterile sites.

    • Unhelpful for distinguishing bacteria in cases like gastroenteritis (intestinal infections).

  • Outcome: Stained slide confirms bacterial presence; further testing (gram stain, biochemical testing) reveals E. coli infection (gram-negative rods).