Microbiology: Culture, Preservation, Growth, and More

Posted on Dec 17, 2024 in Biology

Types of Culture Media

Culture media provide nutrients to support the growth of microorganisms. Their types depend on physical consistency, composition, and intended purpose:

Based on Consistency:

• Liquid Media:
  • Lacks agar and is entirely liquid. Used for growing large bacterial populations.
  • Example: Nutrient Broth, often used for maintaining stock cultures.
• Solid Media:
  • Contains 1.5–2% agar to solidify the medium. Solid media allow for bacterial isolation into distinct colonies.
  • Example: Blood Agar, which supports the growth of fastidious organisms and helps in detecting hemolysis.
• Semi-Solid Media:
  • Contains 0.3–0.5% agar, which gives it a gel-like consistency. Often used to assess motility and growth of anaerobes.
  • Example: Motility Medium, useful for determining whether bacteria are flagellated.

Based on Composition:

• Synthetic/Defined Media:
  • All components and concentrations are precisely known. Useful in research for studying specific metabolic pathways.
  • Example: Minimal Media, which contains salts and a carbon source.
• Complex/Undefined Media:
  • Contains complex ingredients (e.g., yeast extract, peptone) where exact composition is unknown.
  • Example: Tryptic Soy Agar, commonly used for general bacterial growth.

Based on Function:

• Selective Media:
  • Encourages the growth of specific microbes while suppressing others.
  • Example: MacConkey Agar, which promotes Gram-negative bacteria by inhibiting Gram-positive bacteria with bile salts.
• Differential Media:
  • Allows visual differentiation of microbes based on metabolic activity.
  • Example: Eosin Methylene Blue (EMB) Agar, where lactose fermenters produce colored colonies.
• Enrichment Media:
  • Contains nutrients or conditions that favor the growth of a particular organism.
  • Example: Selenite F Broth, used for enriching Salmonella from stool samples.
• Transport Media:
  • Maintains bacterial viability during transport but does not allow multiplication.
  • Example: Stuart’s Medium, used for transporting throat swabs.

Preservation of Bacteria

Bacteria can be preserved for both short- and long-term purposes using various methods:

Short-Term Preservation:

• Refrigeration (4°C):
  • Bacteria are stored on agar slants or in broth. This method preserves viability for weeks.

Long-Term Preservation:

• Deep-Freezing (-20°C to -80°C):
  • Cells are suspended in a cryoprotectant solution (e.g., glycerol or DMSO) to prevent ice crystal formation.
• Lyophilization (Freeze-Drying):
  • Cells are frozen and then dehydrated under vacuum, producing a powder that can be rehydrated for culture. Used for commercial and research purposes.

Growth Curve of Bacteria

When bacteria are cultured in a closed system, their growth follows a predictable pattern:

1. Lag Phase:

   • Cells adapt to the new environment by synthesizing enzymes and molecules needed for growth.
   • No increase in cell numbers occurs.

2. Log (Exponential) Phase:

   • Rapid division occurs as nutrients are abundant, and conditions are optimal.
   • Growth rate is constant, and this phase is used to calculate the doubling time of the bacteria.

3. Stationary Phase:

   • Growth slows as nutrients are depleted and waste accumulates.
   • The population reaches a dynamic equilibrium, where growth rate equals the death rate.

4. Death Phase:

   • Cells die at an exponential rate due to lack of nutrients and toxic waste accumulation.
   • Some species form spores to survive harsh conditions.

Sterilization

Sterilization is the process of eliminating all microorganisms, including spores, from an object or environment.

Methods of Sterilization:

1. Physical Methods:
   • Heat Sterilization:
     • Moist Heat: Autoclaving at 121°C for 15 minutes under 15 psi pressure effectively kills all microbes and spores.
     • Dry Heat: Oven sterilization at 170°C for 2 hours; used for glassware and powders.
   • Filtration:
     • Used for heat-sensitive liquids. Filters with pore sizes of 0.22 µm or smaller can trap bacteria and some viruses.
   • Radiation:
     • UV Radiation: Sterilizes surfaces by damaging microbial DNA.
     • Gamma Radiation: Used for medical devices and food preservation.
2. Chemical Methods:
   • Ethylene Oxide Gas: Sterilizes heat-sensitive equipment like plastic syringes.
   • Disinfectants: Alcohol, chlorine, and phenol are commonly used for surface sterilization.

Staining Techniques

Gram Staining:

A differential stain that distinguishes between Gram-positive and Gram-negative bacteria based on cell wall structure.

1. Primary Stain: Crystal violet colors all cells purple.
2. Mordant: Iodine forms a complex with crystal violet in Gram-positive cells.
3. Decolorizer: Alcohol or acetone removes the dye from Gram-negative cells.
4. Counterstain: Safranin stains Gram-negative cells pink, while Gram-positive remain purple.

Acid-Fast Staining:

Used for bacteria like Mycobacterium that have waxy, lipid-rich cell walls.

1. Primary Stain: Carbol fuchsin penetrates the waxy layer.
2. Decolorization: Acid-alcohol removes the stain from non-acid-fast cells.
3. Counterstain: Methylene blue stains non-acid-fast cells blue.

Louis Pasteur’s Contributions

• Disproved Spontaneous Generation:

  • His swan-neck flask experiment demonstrated that microorganisms in the air cause contamination.

• Pasteurization:

  • Developed a method to heat liquids (e.g., milk, wine) to destroy pathogenic microbes while preserving flavor.

• Vaccines:

  • Developed the first vaccines for rabies and anthrax.

• Germ Theory of Disease:

  • Proved that specific microbes cause specific diseases, paving the way for microbiology.

Prokaryotic vs. Eukaryotic Cells

Spontaneous Generation

Spontaneous generation is an archaic biological theory suggesting that living organisms can arise from non-living matter under certain conditions. For instance, it was once believed that maggots spontaneously appeared in decaying meat or that mice could form from grain and straw.

Impact of Pasteur’s Work

• Pasteur’s experiments laid the foundation for the Germ Theory of Disease, which states that microorganisms are the cause of many diseases.
• This work debunked centuries-old beliefs and marked the beginning of modern microbiology.

Classification of Fungi

Fungi are classified into major groups based on their reproductive structures and spore types:

1. Zygomycota:

• Known as bread molds.
• Characterized by the production of thick-walled sexual spores called zygospores.
• Example: Rhizopus stolonifer (black bread mold).

2. Ascomycota (Sac Fungi):

• Largest fungal group, includes molds, yeasts, and morels.
• Sexual reproduction involves the formation of spores inside sac-like structures called asci.
• Produce asexual spores called conidia.
• Examples:
  • Aspergillus (used in fermentation).
  • Candida albicans (causes candidiasis).

3. Basidiomycota (Club Fungi):

• Known for their club-shaped reproductive structures called basidia, where sexual spores (basidiospores) are produced.
• Includes mushrooms, puffballs, and rusts.
• Example: Agaricus bisporus (common mushroom).

4. Deuteromycota (Imperfect Fungi):

• No known sexual stage in their life cycle.
• Often reclassified into other groups when sexual stages are discovered.
• Example: Penicillium (source of penicillin).

5. Chytridiomycota:

• Primitive fungi with motile spores (zoospores) containing flagella.
• Example: Batrachochytrium dendrobatidis (affects amphibians).

Reproduction in Fungi

Fungi reproduce by both asexual and sexual means, often depending on environmental conditions.

Asexual Reproduction:

Occurs during favorable conditions for rapid propagation.

1. Fragmentation:

   • Mycelium breaks into fragments, and each fragment develops into a new organism.
   • Example: Rhizopus.

2. Budding:

   • Common in yeasts. A small outgrowth (bud) forms on the parent cell, enlarges, and detaches as a new cell.
   • Example: Saccharomyces cerevisiae.

3. Asexual Spore Formation:

   • Sporangiospores:
     • Formed within a sporangium (sac-like structure) at the tip of a hypha.
     • Example: Rhizopus.
   • Conidia:
     • Free spores that form at the tips of specialized hyphae called conidiophores.
     • Example: Aspergillus.

Sexual Reproduction:

Occurs under adverse conditions to promote genetic diversity.

1. Plasmogamy:

   • Fusion of cytoplasm from two parent cells.

2. Karyogamy:

   • Fusion of nuclei to form a diploid zygote.

3. Meiosis:

   • Produces haploid spores from the diploid zygote, which germinate into new fungi.

Types of Sexual Spores:

• Zygospores: Formed by Zygomycota during plasmogamy.
• Ascospores: Produced in asci by Ascomycota.
• Basidiospores: Produced on basidia by Basidiomycota.

Importance of Fungi

Ecological Roles:

• Decomposers: Break down organic material, recycling nutrients in ecosystems.
• Symbiotic Associations:
  • Mycorrhizae: Fungi and plant roots exchange nutrients.
  • Lichens: Symbiosis between fungi and algae or cyanobacteria.

Economic and Medical Roles:

• Food Production:

  • Yeasts are used in fermentation to produce bread, beer, and wine.
  • Molds are used in cheese production (e.g., Penicillium roqueforti in blue cheese).

• Antibiotics:

  • Penicillium produces penicillin, the first widely-used antibiotic.

• Pathogens:

  • Cause diseases in plants (Puccinia causing wheat rust) and humans (Candida causing candidiasis).

Key Examples of Fungi

Fungi are essential to life on Earth, with their diverse structures and reproductive strategies enabling them to thrive in almost any environment. Let me know if you’d like more details on specific fungi or processes!