Unit 3: Microbiology

1. Classification of Microorganisms

Microorganisms are organisms that are too small to be seen with the naked eye. They are classified into a hierarchical system based on their evolutionary relationships. The modern system is the Three-Domain System, which divides cellular life into Archaea, Bacteria, and Eukarya.

1.1. Prokaryotes vs. Eukaryotes

This is the most fundamental division in cellular organization.

1.2. Major Groups of Microorganisms

1. Bacteria (Domain: Bacteria)

   • Characteristics: Unicellular prokaryotes. Cell walls contain peptidoglycan. Diverse metabolic capabilities (photosynthetic, heterotrophic, chemotrophic).
   • Morphology (Shape):
     • Coccus: Spherical (e.g., Staphylococcus aureus).
     • Bacillus: Rod-shaped (e.g., Escherichia coli).
     • Spirillum/Spirochete: Spiral (e.g., Treponema pallidum).
   • Relevance: Pathogens, decomposers, industrial applications (fermentation, bioremediation), part of normal human flora.

2. Archaea (Domain: Archaea)

   • Characteristics: Unicellular prokaryotes. Cell walls lack peptidoglycan. Often found in extreme environments (extremophiles), such as hot springs (thermophiles), high-salt environments (halophiles), and anaerobic environments (methanogens).
   • Relevance: Not known to cause disease in humans. Important in nutrient cycles (e.g., carbon, nitrogen).

3. Fungi (Domain: Eukarya)

   • Characteristics: Eukaryotic, heterotrophic organisms with chitin in their cell walls.
   • Types:
     • Yeasts: Unicellular (e.g., Saccharomyces cerevisiae - baker's yeast).
     • Molds: Multicellular, form filamentous structures called hyphae (e.g., Penicillium chrysogenum).
   • Relevance: Decomposers, food production (bread, beer, cheese), antibiotic production, plant and animal pathogens.

4. Protozoa (Domain: Eukarya)

   • Characteristics: Unicellular eukaryotes, often motile. "Animal-like" protists.
   • Classification by Motility:
     • Amoebas: Move using pseudopods.
     • Ciliates: Move using cilia (e.g., Paramecium).
     • Flagellates: Move using flagella (e.g., Giardia lamblia).
   • Relevance: Important in aquatic food webs, some are major pathogens (e.g., Plasmodium - malaria).

5. Algae (Domain: Eukarya)

   • Characteristics: Unicellular or multicellular eukaryotes. "Plant-like" protists. Capable of photosynthesis. Cell walls typically contain cellulose.
   • Relevance: Major producers of oxygen and organic matter in aquatic environments. Source of agar and other useful products. Algal blooms can be toxic.

6. Viruses (Acellular)

   • Characteristics: Not considered living organisms as they are acellular and metabolically inert outside a host cell. Obligate intracellular parasites.
   • Structure: Consist of genetic material (DNA or RNA) enclosed in a protein coat called a capsid. Some have an outer lipid envelope.
   • Relevance: Cause a wide range of diseases in all types of life (e.g., influenza, HIV, bacteriophages). Used in genetic engineering as vectors.

2. Laboratory Techniques for Microbial Culture

2.1. Culture Media

Culture media provide the necessary nutrients for microbial growth in a laboratory setting.

• Nutrient Broth: A liquid medium used for growing large quantities of bacteria. It contains peptone (a protein digest), beef extract, and water. Growth is observed as turbidity (cloudiness).
• Nutrient Agar: A solid medium created by adding agar (a solidifying agent derived from seaweed) to nutrient broth.
  • Properties of Agar: Melts at ~95°C and solidifies at ~40°C. It is not degraded by most microorganisms.
  • Use: Allows for the growth of bacteria as visible, discrete colonies on its surface.

2.2. Aseptic Technique

A set of practices used to prevent contamination of cultures, sterile media, and the lab environment. Key practices include flaming loops, working near a Bunsen burner flame, and keeping plates/tubes covered.

2.3. Serial Dilution

A stepwise dilution of a substance in solution. In microbiology, it is used to reduce the concentration of microbial cells in a sample to a countable level.

• Purpose: To obtain a sample dilute enough to yield a countable number of colonies (typically 30-300) on an agar plate.
• Procedure:
  1. A series of tubes (dilution blanks), each containing a known volume of sterile diluent (e.g., 9 mL of water or saline), are prepared.
  2. A known volume of the original sample (e.g., 1 mL) is transferred to the first tube. This is a 1:10 or 10⁻¹ dilution.
  3. The first tube is mixed thoroughly. 1 mL is then transferred from this tube to the second tube, creating a 1:100 or 10⁻² dilution.
  4. This process is repeated for the desired number of dilutions.

2.4. Plating Techniques for Isolation and Enumeration

1. Streak Plating

   • Purpose: To isolate pure cultures from a mixed population. The goal is to obtain single, well-separated colonies, each arising from a single cell.
   • Method (Quadrant Streak):
     1. A sterile inoculating loop is used to pick up a sample of the mixed culture.
     2. The sample is streaked over a small area (Quadrant 1) of the agar plate.
     3. The loop is sterilized by flaming and allowed to cool.
     4. The loop is dragged from the end of the first streak into a new area (Quadrant 2).
     5. The loop is re-sterilized and the process is repeated for Quadrants 3 and 4.
   • Result: The cell density decreases with each streak, and by the final quadrant, individual cells are deposited far enough apart to grow into isolated colonies.

2. Spread Plating

   • Purpose: To enumerate viable bacteria in a sample and obtain isolated colonies.
   • Method:
     1. A small, known volume (typically 0.1 mL or 100 µL) of a liquid sample (often from a serial dilution) is pipetted onto the surface of a solidified agar plate.
     2. A sterile, L-shaped glass or plastic spreader ("hockey stick") is used to evenly distribute the inoculum across the entire surface of the agar.
   • Result: Colonies grow only on the surface of the agar. It is good for organisms that are sensitive to heat.

3. Pour Plating

   • Purpose: To enumerate viable bacteria in a sample.
   • Method:
     1. A known volume (typically 1.0 mL) of a liquid sample is pipetted into a sterile, empty Petri dish.
     2. Molten, cooled agar (~45-50°C) is poured into the Petri dish over the sample.
     3. The plate is gently swirled to mix the sample with the agar.
     4. The agar is allowed to solidify.
   • Result: Colonies grow both on the surface and embedded within the agar (subsurface colonies). This method is useful for growing microaerophiles or facultative anaerobes that grow better in low-oxygen conditions. The main disadvantage is that heat-sensitive organisms may be killed by the molten agar.

3. Techniques for Enumeration of Bacteria

Enumeration is the process of counting the number of cells in a sample.

3.1. Direct Methods (Counting Cells)

1. Viable Plate Count (Standard Plate Count)

   • Principle: Based on the assumption that one living cell will give rise to one visible colony. It counts only viable (living) cells.
   • Method: Uses serial dilution combined with either spread plating or pour plating.
   • Calculation:
     • Count the number of colonies on a plate that has between 30 and 300 colonies. This range provides statistical accuracy.
     • The concentration of cells in the original sample is calculated as Colony Forming Units (CFU) per milliliter (CFU/mL).
       
       TEXT

               CFU/mL = (Number of Colonies) / (Volume Plated in mL * Dilution Factor)
               

     • Example: You count 150 colonies on a spread plate where you plated 0.1 mL from a 10⁻⁴ dilution tube.
       • CFU/mL = 150 / (0.1 mL * 10⁻⁴) = 150 / 10⁻⁵ = 1.5 x 10⁷ CFU/mL

2. Direct Microscopic Count

   • Principle: A known volume of sample is placed on a specialized slide (e.g., Petroff-Hausser counting chamber) with a grid of known area and depth. Cells are counted directly under a microscope.
   • Advantages: Rapid and simple.
   • Disadvantages:
     • Counts both living and dead cells.
     • Not accurate for low-density samples.
     • Small, motile cells can be difficult to count.

3.2. Indirect Methods (Measuring Cell Mass or Activity)

1. Turbidity (Spectrophotometry)

   • Principle: As bacteria grow in a liquid culture, the broth becomes more turbid (cloudy). A spectrophotometer measures this turbidity by shining a beam of light through the sample and measuring how much light is scattered or absorbed. The measurement is called Optical Density (OD) or Absorbance.
   • Relationship: Higher cell concentration leads to higher turbidity and thus a higher OD reading.
   • Advantages: Very rapid, non-destructive, and excellent for monitoring growth over time.
   • Disadvantages:
     • Does not distinguish between living and dead cells.
     • Requires a high concentration of cells to be detectable.
     • Requires a standard curve (correlating OD to CFU/mL) for accurate quantification.

2. Dry Weight

   • Principle: The culture is centrifuged to pellet the cells, which are then washed, dried in an oven, and weighed.
   • Use: Best for filamentous organisms like molds, where direct counting is difficult.
   • Disadvantages: Time-consuming and counts both living and dead cells.

4. Growth Kinetics

Bacterial growth refers to an increase in the number of cells, not the size of individual cells. Most bacteria reproduce by binary fission, where one cell divides to form two identical daughter cells.

4.1. The Bacterial Growth Curve

When bacteria are inoculated into a fresh liquid medium and incubated, they exhibit a characteristic pattern of growth with four distinct phases.

1. Lag Phase:

   • Description: Period of adaptation. There is no increase in cell number.
   • Cellular Activity: Cells are metabolically active, synthesizing new enzymes, ATP, and other molecules needed for growth in the new environment. The length of this phase depends on the condition of the inoculum and the nature of the new medium.

2. Log (Exponential) Phase:

   • Description: Cells divide at a constant, maximum rate. The population doubles with each generation.
   • Cellular Activity: Cells are healthiest and most uniform. They are most sensitive to antibiotics and adverse conditions during this phase.
   • Kinetics: Growth is exponential. A plot of the natural logarithm of cell number vs. time is a straight line.

3. Stationary Phase:

   • Description: The population growth rate slows to zero; the rate of cell division equals the rate of cell death.
   • Causes: Depletion of essential nutrients, accumulation of toxic waste products (e.g., acids), changes in pH, or oxygen limitation.

4. Death (Decline) Phase:

   • Description: The rate of cell death exceeds the rate of cell division. The number of viable cells decreases exponentially.
   • Causes: Complete exhaustion of nutrients and lethal accumulation of toxins.

4.2. Mathematical Expressions of Growth

• Generation Time (g) or Doubling Time (t_d): The time required for a population to double in number.
• Number of Generations (n): The number of doublings that have occurred.

The relationship between the initial number of cells (N₀), the final number of cells (N), and the number of generations (n) is:

N = N₀ * 2ⁿ

To solve for the number of generations (n):

log(N) = log(N₀) + n * log(2)
n = (log(N) - log(N₀)) / log(2)

The generation time (g) can then be calculated if the total time (t) is known:

g = t / n

• Specific Growth Rate (μ): A measure of the number of divisions per cell per unit time. It is the slope of the line when ln(N) is plotted against time (t) during the exponential phase. It is inversely related to generation time.
  
  TEXT

  μ = ln(2) / g   (or  μ ≈ 0.693 / g)
  

5. Food Spoilage and Preservation

5.1. Concept of Food Spoilage

Food spoilage is any change in the visual appearance, odor, or taste of a food product that makes it unacceptable to the consumer. It is primarily caused by the growth and metabolic activity of microorganisms (bacteria, yeasts, molds).

Factors Affecting Microbial Growth in Food:

• Intrinsic Factors (Properties of the food itself):
  • Water Activity (a_w): The amount of free water available for microbial use. Most spoilage bacteria require a_w > 0.9.
  • pH: Most microbes prefer a neutral pH (~7.0). Acidic foods (pH < 4.6) like fruits are more resistant to bacterial spoilage but susceptible to fungal spoilage.
  • Nutrient Content: The presence of proteins, carbohydrates, and fats influences which microbes can grow.
  • Antimicrobial Compounds: Some foods contain natural antimicrobials (e.g., essential oils in spices).
• Extrinsic Factors (Storage environment):
  • Temperature: Temperature is a critical factor. Different microbes have different optimal growth temperatures (psychrophiles, mesophiles, thermophiles).
  • Atmosphere: The presence or absence of oxygen determines whether aerobic, anaerobic, or facultative organisms will grow.
  • Relative Humidity: Affects the water activity on food surfaces.

5.2. Food Preservation Techniques

Preservation techniques aim to control microbial growth by manipulating intrinsic and extrinsic factors to slow or stop spoilage and kill pathogens.

1. High-Temperature Methods (Thermal Processing)

   • Principle: Heat denatures essential proteins and enzymes, killing microorganisms.
   • Pasteurization: Mild heat treatment that kills most pathogens and reduces the number of spoilage organisms. It is not sterilization.
     • HTST (High-Temperature Short-Time): 72°C for 15 seconds (e.g., milk).
     • UHT (Ultra-High Temperature): 140-150°C for 1-3 seconds. Results in a sterile product with a long shelf life.
   • Sterilization (Canning): A severe heat treatment (e.g., 121°C for 15 minutes) that destroys all microorganisms, including heat-resistant bacterial endospores (e.g., Clostridium botulinum).

2. Low-Temperature Methods

   • Principle: Low temperatures slow down or inhibit enzymatic reactions and microbial growth.
   • Refrigeration (0-7°C): Slows the growth of mesophilic spoilage organisms. Psychrotrophs can still grow slowly.
   • Freezing (< -18°C): Stops microbial growth by making water unavailable (frozen). Does not necessarily kill all microbes; they can resume growth upon thawing.

3. Reduction of Water Activity (Dehydration)

   • Principle: Removing water inhibits microbial growth as water is essential for metabolism.
   • Drying: Sun drying, oven drying, freeze-drying.
   • Adding Solutes (Salt or Sugar): Curing meats with salt or preserving fruits with sugar creates a hypertonic environment. This causes water to leave microbial cells via osmosis (plasmolysis), inhibiting their growth.

4. Chemical Preservation

   • Principle: Adding chemicals that are antimicrobial or alter the environment to be unfavorable for microbial growth.
   • Acidification (Lowering pH): Adding acids like vinegar (acetic acid) or lactic acid (from fermentation) directly inhibits most bacterial growth. Example: Pickling.
   • Adding Preservatives:
     • Nitrates/Nitrites: Used in cured meats to prevent the growth of Clostridium botulinum.
     • Sulfites: Used in wine and dried fruits to inhibit fungi.
     • Benzoates/Sorbates: Used in acidic foods like soft drinks and jams to inhibit yeasts and molds.

5. Irradiation

   • Principle: Uses ionizing radiation (gamma rays, X-rays) to damage microbial DNA, leading to cell death. Can be used to sterilize spices, meats, and produce without significant heat.

6. Modified Atmosphere Packaging (MAP)

   • Principle: The air in a food package is replaced with a specific gas mixture (e.g., low O₂, high CO₂, high N₂) to slow spoilage.
   • Example: High CO₂ levels inhibit the growth of aerobic bacteria and molds. Removing O₂ prevents the growth of obligate aerobes. Used for fresh meats, salads, and pasta.