Microbiology Course Notes

Microbiology Overview

General Information

  • Course Title: Microbio310 General Microbiology
  • Instructor: Dr. Verena Carvalho, UMass Amherst

Key Subjects Covered

  • Culturing, media, microbial growth, and its control

Module 3: Bacteria in Research and Industry

Topics Covered

  1. Growing bacteria in the lab
  2. Applications in genetic engineering
  3. Applications in industry and bioremediation

Module 3 – 1: Learning Objectives

  • After the lecture, students will be able to:
    1. Outline the design of different growth media and growth conditions.
    2. Discuss what growth in different media and/or under certain conditions tells us about characteristics of microorganisms.
    3. Explain sterile/aseptic technique as a mechanism to control microbial growth.
    4. Define isolation/pure culture of bacteria and understand what constitutes a colony.

Historical Context of Microbiology

Louis Pasteur (1822–1895)

  • Profession: Chemist and microscopist.
  • Contributions:
    • Discovered that alcoholic fermentation is biologically mediated, not just chemically.
    • Developed vaccines for anthrax, cholera, and rabies.
    • Utilized the swan-necked Pasteur flask to disprove spontaneous generation, a common belief until around 1860 that life could arise from nonliving matter.
    • His work led to the establishment of sterilization methods and food preservation techniques, emphasizing the role of microorganisms.

Swan-necked Pasteur Flask

  • Designed to show that microorganisms in the air could contaminate sterile solutions; the curved neck traps dust and bacteria, keeping the liquid sterile unless the flask is tilted.

Robert Koch (1843–1910)

  • Profession: Physician and microbiologist.
  • Contributions:
    • Developed solid media for obtaining pure cultures of microbes.
    • Observed the diversity of cell masses, noting different shapes, colors, and sizes of colonies.
    • Formulated Koch's postulates, which are criteria to establish a causative relationship between a microbe and a disease, effectively linking microbes to infectious diseases.
    • Identified causative agents for anthrax, tuberculosis, and cholera.

Koch's Postulates

  • These are foundational criteria for establishing a link between a specific microbe and a specific disease. There are four criteria:
    1. The microbe must be found in abundance in all organisms suffering from the disease, but should not be found in healthy organisms.
    2. The microbe must be isolated from a diseased organism and grown in pure culture.
    3. The cultured microbe should cause disease when introduced into a healthy organism.
    4. The microbe must be re-isolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.

Control of Microorganisms

Koch & Pasteur's Contributions

  1. Invented growth media and methods for sterilization (turbidity control).
  2. Established inoculation methods for controlled growth of microorganisms.
  3. Enabled study of colonies created under controlled growth conditions.

Growing Bacteria in the Laboratory

Sterile Techniques

  • Procedures and special equipment are crucial to avoid contamination:
    1. Prevent contamination from the external environment into cultures or experiments.
    2. Prevent contamination from bacterial cultures to external environments, mitigating infection risks.
  • Common Tools:
    • Bunsen burner
    • Inoculation loop

Sterilization Methods

  • Autoclaving: Uses pressurized saturated steam at 121ext°C121^{ ext{°C}} for 30-60 minutes to destroy all biomolecules, achieving COMPLETE sterilization.
  • Pasteurization: Heats liquids to 75ext°C75^{ ext{°C}} for 30 seconds to destroy most microorganisms but not endospores.
  • Filtration: Utilized for sterilizing liquids or air; some very small particles remain.
  • Sanitation Agents:
    • Alcohols (ethyl and isopropyl): Acts as antiseptics for skin.
    • Sodium hypochlorite (bleach): A harsh chemical disinfectant.
    • Radiation (e.g., UV): Effective but does not reach behind objects.

Growth Media for Microbiological Studies

Types of Growth Media

  • Sterile Nutrient Solutions: Liquid (broth) or with solidifying agents (agar), providing macronutrients (C, N, P, S) and micronutrients (minerals, metals, vitamins).
Media Composition
  1. Defined Media:
    • Exact chemical composition of nutrients is known.
    • Used for specific physiological tests requiring known nutritional requirements.
  2. Complex/Undefined Media:
    • Composed of boiled or pre-digested products with unknown nutrient compositions.
    • High nutrient concentration fosters growth of many species, not representative of natural conditions.
    • Examples include adding specific amounts (e.g., 5extg5 ext{ g} of glucose or 2extg2 ext{ g} of lactose).

Selective & Differential Media

Media Definitions

  • Selective Media: Contains additives to inhibit growth of specific, unwanted microbes using agents like bile salts.
  • Differential Media: Incorporates substances to visualize biochemical reactions, leading to color changes (e.g., using pH-sensitive dyes).

Example: Mannitol Salt Agar (MSA)

  • Selective: High salt content (6.5%) inhibits many microbes.
  • Differential: If mannitol is fermented, it results in acid production changing the color of the pH-sensitive dye phenol red from red to yellow.
  • Organisms Observed:
    • Staphylococcus aureus: Grows, leading to yellowing of the medium.
    • Serratia marcescens: Shows no growth, maintaining red color of medium.

Obtaining Pure Cultures

Definition: Pure Culture

  • Descendants of a single cell; obtained through processes such as dilution to extinction.
  • A colony is a visible mass of cells originating from one mother cell, becoming noticeable with increased cell density.
  • Examples of mixed/natural samples include pond water, wastewater, saliva, and wound swabs.

Techniques

  • Streaking for Isolation: Technique to distribute individual cells to isolate a single colony from a mixed culture.
  • Re-streaking: Picking one isolated colony type to achieve a pure culture containing only one bacterial species.

Bacterial Batch Culture Growth Curve

Phases of Growth Curve

  1. Lag Phase: Initial adjustment phase for cells to adapt to the new environment.
  2. Exponential Phase: Cells divide at regular intervals, with optimal function of enzymes and biological processes.
  3. Stationary Phase: Growth arrests, but energy and metabolic processes may continue. Nutrient levels decline, and toxic waste accumulates, applying stress on cells.
  4. Death Phase: Cells begin to die due to accumulation of waste products and decreasing nutrient concentrations.

Environmental Factors Affecting Bacterial Growth

Temperature Testing

  • Human pathogens typically categorized as mesophiles; body temperature increases can be lethal for cells (e.g., fever).

Oxygen Tolerance Testing

  1. Obligate Aerobes: Require oxygen, thriving at full atmospheric levels (20% oxygen).
  2. Obligate Anaerobes: Harmed or killed by oxygen exposure.
  3. Facultative Anaerobes: Prefer oxygen but can grow in the absence of it through alternative metabolic pathways.
  4. Microaerophiles: Utilize oxygen only at lower-than-atmospheric levels due to sensitivity.
  5. Aerotolerant: Do not need oxygen but can tolerate its presence; oxygen forms reactive oxygen species.