Microbiology Lecture: Bacterial Morphology and Growth

Principles of Microbial Growth

• Definition: Growth refers to an increase in the number of cells, not the size of an individual cell.

• Binary Fission: The process of asexual reproduction where a cell increases its volume, duplicates its DNA, and splits into two identical daughter cells.

• Exponential Growth: Cell division is exponential ($1$ to $2$, $2$ to $4$, etc.). Starting with $10$ cells with a $20$-minute generation time results in over $40,000$ cells in $4$ hours ($12$ generations).

• Generation Time: The time it takes for a population to double. Varies by species and conditions:

  • $E. coli$: $20$ minutes (optimum).

  • $Mycobacterium$ $tuberculosis$: $12$ hours.

  • $Treponema$ $pallidum$: $33$ hours.

  • $Mycobacterium$ $leprae$: At least $10$ days.

• Growth Media:

  • Broth: Liquid medium.

  • Agar: A solidifying agent extracted from marine red algae. It liquefies at $95\,^{\circ}C$ and solidifies at $45\,^{\circ}C$. It remains solid at $37\,^{\circ}C$, the standard incubation temperature.

• Pure Culture: A population derived from a single cell (clones). Obtained using the streak plate method, which dilutes bacteria across four quadrants until isolated colonies form.

The Bacterial Growth Curve

Occurs in a closed system (batch culture) with five stages:

1. Lag Phase: No increase in cell number; cells are active, synthesizing proteins/DNA and increasing in size to prepare for division.

2. Log (Exponential) Phase: Cells divide at a constant, rapid rate. Cells are most sensitive to antibiotics (like penicillin) during this phase.

3. Stationary Phase: Population stabilizes. Nutrient depletion and waste buildup lead to equal rates of cell division and cell death.

4. Death Phase: Cells die off at an exponential rate.

5. Phase of Prolonged Decline: A small fraction of cells survive for weeks, months, or even a year by scavenging nutrients from dead cells.

Environmental Factors Influencing Growth

Temperature Groups

• Psychrophile: $-5\,^{\circ}C$ to $15\,^{\circ}C$ (Arctic/Antarctic).

• Psychrotroph: $20\,^{\circ}C$ to $30\,^{\circ}C$ (important in refrigerator food spoilage).

• Mesophile: $25\,^{\circ}C$ to $45\,^{\circ}C$ (includes most human pathogens; optimum is $37\,^{\circ}C$).

• Thermophile: $45\,^{\circ}C$ to $70\,^{\circ}C$ (hot springs).

• Hyperthermophile: $70\,^{\circ}C$ to over $100\,^{\circ}C$ (hydrothermal vents; mostly Archaea).

Oxygen Requirements

• Tolerance Mechanisms: To live with oxygen, cells need enzymes like super-oxide dismutase ($SOD$) to neutralize super-oxide radicals and catalase to neutralize hydrogen peroxide.

• Obligate Aerobes: Absolute requirement for $O_2$; grow at the top of the tube.

• Facultative Anaerobes: Prefer $O_2$ (grow more at top) but can grow without it (grow throughout) using fermentation or anaerobic respiration.

• Obligate Anaerobes: Killed by $O_2$ because they lack $SOD$ and catalase; grow only at the bottom.

• Microaerophiles: Require low concentrations of $O_2$ ($2\%$ to $10\%$); grow in the middle of the tube.

• Aerotolerant Anaerobes: Indifferent to $O_2$; they do not use it but are not killed by it. They grow evenly throughout the tube.

Questions & Discussion

• Student Question on Endospore Formation Timing: A student asked about how long it takes for endospores to form.

• Response: The instructor noted it takes several hours and varies by species. In the lab, $Bacillus$ $megaterium$ forms mostly endospores within $24$ hours, while $Bacillus$ $subtilis$ takes a bit longer.

• Student Question on Growth Curve: A student asked about the transition between phases.

• Response: The instructor emphasized that the lag phase is a period of intense metabolic activity despite the lack of cell division, and the log phase is the period where antibiotics like penicillin exert the most effect.

Environmental Factors Affecting Growth: pH and Water Availability

• The Role of pH in Bacterial Growth:

  • Every bacterium has a specific, narrow pH range within which it can survive.

  • Optimum pH: Similar to temperature, there is a specific pH level at which a bacterium grows best.

  • Internal Cell pH: Regardless of the external environmental pH, the internal pH of the cell must remain near neutral ($\approx 7$) to ensure enzyme functionality.

• Categories Based on pH Preference:

  • Neutrophiles: Bacteria that thrive in surroundings with a neutral pH, typically between $5$ and $8$. Their growth optimum is around $7$.

  • Acidophiles: Bacteria that grow best in acidic environments with a pH below $5.5$. Some can survive at a pH of $0$. One specific organism is noted to grow at a pH of less than $1$.

  • Alkaliphiles: Bacteria that grow best at a basic pH, specifically above $8.5$. Some species can grow in environments up to pH $14$. Even in these basic extremes, the internal pH of the cell remains near neutral.

• Water Availability and Osmotic Pressure:

  • All microorganisms require water to grow as biological processes are water-based.

  • Plasmolysis: Occurs when bacteria are in a high-salt (hypertonic) environment where the concentration of salt is higher outside the cell than inside.

    • Water moves toward the higher solute concentration, causing water to leave the cell.

    • The cytoplasm shrinks, and the cytoplasmic membrane pulls away from the cell wall.

    • This shrinkage inhibits functions like reproduction and leads to cell death.

  • Osmotic Lysis: The opposite of plasmolysis, occurring when the solute concentration is higher inside the cell than outside. Water enters the cell, causing it to expand and potentially burst if the cell wall is weak.

• Microbes in High-Salt Environments:

  • Halotolerance: The ability of certain microorganisms to withstand high salt concentrations without requiring them for growth.

    • Examples: Staphylococcus aureus and Staphylococcus epidermidis.

    • These can tolerate salt concentrations up to $10\%$. This allows them to survive on human skin, which becomes salty during sweating.

  • Halophiles: Salt-loving organisms that require high salt concentrations to survive.

    • Marine Bacteria: Require a concentration of $3\%$ (the average salt concentration of the ocean).

    • Extreme Halophiles: Require concentrations of $9\%$ or higher and may die by lysing (bursting) if placed in low-salt environments.

    • Locations: Found in the Dead Sea and the Great Salt Lake in Utah, where shores are often covered in saturated salt rather than sand.

Nutritional Factors and Required Elements

• Prokaryotic Nutrient Requirements:

  • Essential elements are required to build macromolecules including DNA, RNA (nucleic acids), carbohydrates, proteins, and fats.

• Groupings Based on Carbon Source:

  • Autotrophs: Use inorganic carbon specifically in the form of carbon dioxide ($CO_2$) gas.

  • Heterotrophs: Cannot use inorganic carbon; they must use organic compounds like glucose.

• Groupings Based on Energy Source:

  • Phototrophs: Derive energy from sunlight (photosynthetic organisms).

  • Chemotrophs: Derive energy from chemical compounds. Humans and most non-photosynthetic organisms are chemotrophs.

• Trace Elements (Minerals):

  • Required only in very minute amounts for proper enzyme function.

  • Includes: Cobalt, Zinc, Copper, Molybdenum, Manganese, Calcium, and Magnesium.

• Growth Factors:

  • Essential substances that bacteria cannot synthesize themselves and must be provided in the growth medium.

  • Includes specific vitamins, purines, pyrimidines (for DNA/RNA synthesis), and essential amino acids.

  • Fastidious Organisms: Microbes with complicated nutritional requirements that are difficult to replicate in a laboratory setting. Many human pathogens are fastidious.

Laboratory Culture Media and Isolation Techniques

• General Categories of Media:

  • Complex Media: Contains a variety of ingredients (e.g., yeast extract, beef extract) where the exact chemical composition is unknown and varies batch to batch.

    • Examples: Nutrient Agar (NA), Blood Agar, and Chocolate Agar.

  • Chemically Defined Media: Prepared by weighing every specific ingredient; the precise amount of every chemical is known.

    • Used primarily in research for highly controlled experiments.

• Selective and Differential Media:

  • Selective Media: Inhibits the growth of unwanted microorganisms while allowing target microbes to grow.

    • Thayer-Martin Agar: Used to isolate Neisseria gonorrhoeae from genital swabs by inhibiting the normal microbiome.

    • Macombi Agar: Specifically used to isolate gram-negative bacteria; it contains dyes that inhibit gram-positive growth.

  • Differential Media: Contains substances that bacteria act upon to produce a recognizable change.

    • Blood Agar: Contains $5\%$ sheep's blood. It differentiates bacteria based on their ability to produce the toxin/enzyme hemolysin.

      • Beta (\beta) Hemolysis: Complete lysis of red blood cells, resulting in a transparent, see-through area around the growth.

      • Alpha (\alpha) Hemolysis: Partial lysis that produces a greenish appearance.

      • Gamma (\gamma) Hemolysis: No hemolysis occurs.

    • Macombi Agar (Differential aspect): Contains lactose and a pH indicator.

      • Lactose Positive: Organisms like E. coli ferment lactose, producing acid that turns the growth red.

      • Lactose Negative: Organisms like Pseudomonas aeruginosa cannot break down lactose and appear pale.

Atmospheric Requirements for Growth

• Capnophiles: Require high concentrations of $CO_2$ (higher than atmospheric air).

  • Growth Methods: Historically grown in "candle jars" where a burning candle consumes oxygen and releases $CO_2$. Modern labs use specialized $CO_2$ incubators.

• Microaerophiles: Require lower concentrations of oxygen ($O_2$) than what is present in the atmosphere.

• Anaerobes: Organisms that die in the presence of oxygen and are difficult to grow.

  • Anaerobic Jar: Uses packets to initiate catalysts that replace oxygen with other gases.

  • Reducing Agents: Chemicals like sodium thioglycolate added to liquid media to bind up oxygen.

  • Anaerobic Chamber: An enclosed workspace with sleeves and air-locked doors where all oxygen is flushed out and replaced with other gases; often contains its own internal incubator.

Methods for Measuring Bacterial Growth

• Direct Methods:

  • Direct Microscopic Count: Liquid samples are placed on a special slide (grid) with a known volume. Bacteria are counted under a microscope to estimate total population. It does not distinguish between living and dead cells.

  • Viable Plate Count: Counts only live bacteria that can form colonies on solid agar.

    • Serial Dilutions: Required because cultures often contain too many bacteria to count i directly.

    • Spread Plate vs. Pour Plate: In pour plates, some colonies grow within the agar; in spread plates, they grow on the surface.

    • Units: Expressed as Colony Forming Units per mil ($CFU/mL$) because a colony might arise from a cluster or chain of cells, not just one cell.

    • Counting Range: Plates with $30$ to $300$ colonies are chosen for accuracy. Higher counts are TNTC (Too Numerous To Count).

  • Membrane Filtration: Used for samples with very low bacterial concentrations (e.g., water or food).

    • Liquids are passed through a membrane with pores (typically $0.2\,\mu\text{m}$) that trap bacteria.

    • The membrane is placed on agar to allow trapped cells to grow into colonies.

• Indirect Methods:

  • Turbidity: Measuring the cloudiness of a broth using a spectrophotometer.

    • Absorbance: As bacteria increase, more light is obstructed; absorbance is proportional to the concentration of cells.

  • Total Weight (Biomass): Bacteria are filtered, dried, and weighed. Primarily used for filamentous bacteria that grow in long chains, making colony counts inaccurate.