Lecture Notes: Ubiquity of Microorganisms, Culturing, and Isolating Bacterial Colonies

Introduction

• Instructor: Professor Sims
• Course: Laboratory for the Fundamentals of Microbiology
• Session: 2nd in a series of 10 lab sessions
• Learning Objectives:
  • Perform aseptic techniques
  • Understand the ubiquity of microbes
  • Learn types of media: liquid, broths, solid, agar, slants, deeps, plates
  • Prepare streak plates and isolate bacterial colonies
  • Observe and describe bacterial colony morphologies
  • Understand safety and disposal procedures

Microorganisms and Their Characteristics

• Ubiquity: Microorganisms are found everywhere
• Diversity:
  • Prokaryotes: Simple structure, no membrane-bound nucleus, unicellular
  • Eukaryotes: Complex structure, membrane-bound nucleus, can be unicellular or multicellular
  • Viruses: Simple structure, require a host to multiply
• Evolution: Microorganisms have evolved over 4 billion years

Experiment 1: Illustrating Microbial Ubiquity

• Objective: Test bacterial contamination on two inanimate objects within the lab area
• Procedure:
  • Select two objects (e.g., lab bench, door handle)
  • Swab and inoculate nutrient agar plate, divide into two sections
  • Incubate and hypothesize on bacterial growth/species

Bacterial Culturing Guidelines

• Growth Conditions:
  • Neutrophiles: Grow at pH 7
  • Acidophiles: Grow at pH < 7
  • Alkalophiles: Grow at pH 8-11
  • Mesophiles: Optimal growth at 20-45°C, include human pathogens
  • Psychotropes: Grow at 4-25°C
  • Thermophiles: Grow at higher temperatures
  • Hyperthermophiles: Survive extreme heat, e.g., 80-110°C

Oxygen Requirements of Bacteria

• Obligate Aerobes: Require oxygen
• Obligate Anaerobes: Killed by oxygen
• Facultative Anaerobes: Can grow with or without oxygen
• Aerotolerant Anaerobes: Indifferent to oxygen
• Microaerophiles: Require low oxygen levels

Types of Media Used in Lab

• Liquid Broths: Quick culture, observe turbidity
• Solid Agar: Used for isolation and counting bacteria
• Agar Slants: Good for aerobic bacteria
• Agar Deeps: Suitable for anaerobic bacteria, testing motility

Experiment 2: Inoculating E. coli

• Objective: Use E. coli to inoculate slants and deeps
• Procedure:
  • Use aseptic technique
  • Sterilize inoculating loop, inoculate agar slants with a fishtail motion, and deeps by stabbing

Aseptic Technique and Safety

• Wear PPE: lab coat, safety glasses, gloves
• Use back incinerator for sterilizing loops
• Keep specimens at arm's length
• Avoid contamination: Cover plates, sterilize tools, avoid sneezing/coughing near samples

Experiment 3: Isolating Bacterial Colonies

• Objective: Isolate colonies from a mixed culture
• Method:
  • Use streaking technique
  • Sterilize loop between streaks
  • Aim for isolated colonies in third or fourth quadrant

Observations and Interpretations

• Experiment 1: Compare bacterial growth on objects A and B
• Experiment 2: Check E. coli growth on slants and deeps
• Experiment 3: Evaluate isolation success and colony morphology

Conclusion

• Review reading materials
• Check course resources for further studies
• Leave questions for clarification

Additional Notes

• Look up E. coli oxygen requirements and motility (flagella/cilia)
• Practice observing and describing colony morphologies in lab
• Ensure proper closure and cleanup of equipment after lab sessions