BIO 245 Lecture 8: Culturing Bacteria II - Comprehensive Notes

BIO 245 Lecture 8: Culturing Bacteria II

Exam Policies and Logistics

• Exam 1 Countdown: $2$ days until September $18$, $2025$ (Lecture on September $16$, $2025$).

Isolation

• Goal: To obtain colonies of a pure culture of a single organism.

• Types of Isolation Culture Methods:

  • Isolation Streak

  • Pour Plate

  • Spread Plate

The Isolation Streak Method

• Involves specific steps to dilute bacteria across an agar plate, leading to isolated colonies.

Pour Plate Method

• A bacterial suspension is mixed with molten agar (at $45-50^ ext{o}C$).

• The mixture is poured into a plate, allowed to solidify, and then incubated.

• Colonies grow both on the surface and within the agar medium.

Spread Plate Method

• A sample ($0.1 ext{ mL}$) is poured onto a solid medium.

• The sample is spread evenly over the surface using a sterile spreading tool.

• The plate is incubated until bacterial colonies grow on the surface of the medium.

Identification of Unknown Organisms

• Identification relies on a mixture of tests:

  • Microscopy

  • Biochemical tests

  • Molecular tests

Microscopy for Identification

• Gram Status: Differentiates between Gram-positive ($+$, purple) and Gram-negative ($-$, pink) bacteria.

• Morphology: Observes shape (Rods, Cocci) and arrangement.

Biochemical Tests for Identification

• Evaluates metabolic characteristics, such as:

  • Aerotolerance: Ability to grow in the presence or absence of oxygen.

  • Carbon (C) and Nitrogen (N) usage: Determines specific nutrient utilization pathways.

  • Motility: Assesses the organism's ability to move.

  • And other specialized tests.

Molecular ID: $16S$ rRNA Gene

• Discovery: Carl Woese discovered the significance of the $16S$ rRNA gene in $1977$.

• Characteristics making it useful for identification:

  • Functionally constant: Essential for ribosomal structure and function.

  • Universally distributed: Present in all bacteria and archaea.

  • Highly conserved (slow-evolving regions) with variable regions: Allows for comparison across broad phylogenetic ranges, while variable regions resolve differences between similar organisms.

  • Long enough to resolve differences: Provides sufficient sequence information for robust phylogenetic analysis.

Uncultured Microbes

• Challenge: Less than $1$% (sometimes less than $0.001$%) of microbes in any environment can be cultured using standard lab techniques.

• Solution: Enrichment techniques are used to get microbes into culture.

• Enrichment Steps:

  1. Collect your sample: Obtain the environmental sample containing the microbes.

  2. Identify the organism's natural habitat: Understand where your target organism lives.

  3. Mimic natural conditions: Attempt to replicate the specific environmental conditions (e.g., temperature, pH) in the lab.

  4. Inoculate in selective medium/conditions: Design a growth medium and conditions that favor the growth of the desired organism while inhibiting others.

     • Consider both nutrients available (resources) and environmental conditions (e.g., temperature, pH, light).

     • Determine what you want to exclude to promote the growth of the target microbe.

Microbial Nutrition

Most Abundant Molecules and Elements

• Most abundant molecule in any cell: WATER ($70$% of total weight).

• Most abundant organic compounds (by % Dry Weight):

  • Proteins ($50$%)

  • Nucleic acids (RNA: $20$%, DNA: $3$%)

  • Carbohydrates ($10$%)

  • Lipids ($10$%)

  • Miscellaneous ($Not ext{ determined}$)

• Most abundant elements (by % Dry Weight):

  • Carbon ($50$%)

  • Oxygen ($20$%)

  • Nitrogen ($14$%)

  • Hydrogen ($8$%)

  • Phosphorus ($3$%)

  • Sulfur ($1$%)

  • Potassium ($1$%)

  • Sodium ($0.5$%)

  • Calcium ($0.5$%)

  • Magnesium ($0.5$%)

  • Chloride ($0.2$%)

  • Iron ($0.3$%)

  • Trace elements: Manganese, zinc, molybdenum, copper, cobalt.

Importance of Carbon

• Essential component: Forms the backbone of major macromolecules (proteins, nucleic acids, carbohydrates, lipids).

• Key to both cell structure and metabolism.

• Foundation for basic nutritional categories.

Nutrition Types: Carbon Source

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