Week 3 Lab 4 - Ubiquity of microbes, colony morphology, aseptic technique and streak plate isolation method

Lab 4 Overview

  • Title: Ubiquity of Microbes, Colony Morphology, Aseptic Technique, and Streak Plate Isolation Method

Agenda for Lab 4

  • Finish Lab 3B: Microbial chefs will pour media on petri plates.

  • Lab 4 Day 1:

    • 4A: Ubiquity of microorganisms

    • 4B: Aseptic technique

    • 4C: Streak plate isolation method

Lab 3B Overview

  • Focus: Microbial chefs preparing media on plates in Day 2.

Day's Activities

  • 55°C Melting:

    • Remove sterile, melted media from 55°C water bath.

    • Using Aseptic Technique: Pour 20-25 plates, allow cooling and solidification (Instructor demo).

    • Test and record pH once solidified.

    • This media will be used in 4A.

Lab 4A: Ubiquity of Microbes

Microbial Ubiquity

  • Finding Microbes: Microbes are virtually ubiquitous, found in:

    • Air

    • Food

    • Water

    • On human bodies

    • Soil

  • Most microbes benefit daily living.

Factors Influencing Growth on Petri Plates

  • Determined by:

    • Nutrients provided by media type

    • Presence of selective agents

    • Optimal incubation conditions and duration

Today's Activity

  • Sampling Environment for Microbes:

    • Obtain 6 media types from lab 3B per student.

    • Expose plates to well-ventilated areas for 30-60 minutes without lids.

    • Avoid hospitals or healthcare facilities.

    • Incubate at room temperature until growth observed.

    • Use parafilm and zip-lock bags for storage.

    • Refrigerate and bring back to next lab to describe colony morphologies.

Lab 4B: Aseptic Technique

Importance of Aseptic Technique

  • Aseptic techniques prevent contamination in microbiological processes:

    • Protect samples while transferring bacteria.

    • Critical in clinical settings to avoid pathogen transfer.

    • Essential for accurate clinical diagnoses.

Inoculating Instruments

  • Instruments:

    • Inoculating loop and needle for transferring inoculum.

    • Inoculum: Sample moved between media.

  • Sterilization:

    • Instruments sterilized using Bunsen burner flame.

    • Required to sterilize before and after each inoculation.

Aseptic Handling Guidelines

  • Test Tubes:

    • Sterilize the rim before opening.

    • Do not place caps on the benchtop.

  • Petri Plates:

    • Open lids at a 45-degree angle.

    • Avoid leaving plates open to air.

Today's Aseptic Practice

  • Activities:

    • In teams of two, practice labeling tubes and plates with initial, media type, date, sample, incubation temperature, and lab section.

    • Specific inoculation tasks include:

      • Broth to slant inoculation (2 slants).

      • Plate to broth inoculation (2 tubes).

      • Broth to plate inoculation (1 plate).

Lab 4C: Streak Plate Isolation Method

Pure Culture vs. Mixed Culture

  • Pure Cultures:

    • Contain one microbial species.

  • Mixed Cultures:

    • Contain two or more species.

  • Significance:

    • Pure cultures help establish links between microbes and diseases.

    • Critical for identifying species and studying antibiotic susceptibility.

    • Facilitate genome sequencing for treatment understanding.

Streak Plate Isolation Method Explained

  • Purpose:

    • Technique used to separate individual bacteria from mixtures.

  • Process:

    • Spread inoculum on agar surface.

    • Low concentration allows isolated colonies to form from single bacteria or small groups.

  • Colony-Forming Unit (CFU):

    • Term describing that colonies may form from individual cells or clusters.

Today's Streak Plate Practice

  • Activities:

    • Practice streaking for isolation using either Mix A or Mix B.

    • Incubate at 30°C for 24-48 hours.