9.1 How Microbes Grow

Microbial Growth and Reproduction

Microbes multiply rapidly through binary fission, doubling their population over a specific generation time. This growth follows distinct phases that are predictable and measurable. Understanding these phases, along with how microbes form biofilms and communicate, is central to controlling microbial populations in clinical, industrial, and environmental settings.

Generation Time in Binary Fission

Generation time is the time it takes for a microbial population to double through binary fission. A fast-growing species like E. coli can have a generation time as short as 20 minutes under ideal conditions, while slower organisms like Mycobacterium tuberculosis may take 15–20 hours.

You can calculate generation time with this formula:

$G = \frac{t}{n}$

• $G$ = generation time
• $t$ = total elapsed time
• $n$ = number of generations during that time

To find the number of generations:

$n = \frac{\log(N_t) - \log(N_0)}{\log(2)}$

• $N_t$ = final population size
• $N_0$ = initial population size
• You divide by $\log(2)$ because each generation doubles the population (base 2)

Quick example: If you start with 1,000 cells and end with 1,000,000 cells after 200 minutes:

1. Calculate $n$: $n = \frac{\log(1{,}000{,}000) - \log(1{,}000)}{\log(2)} = \frac{6 - 3}{0.301} \approx 10$ generations

2. Calculate $G$: $G = \frac{200}{10} = 20$ minutes per generation

Phases of Microbial Growth Curves

When you plot microbial population size over time (using a log scale for the y-axis), four distinct phases appear:

• Lag phase: Cells are adjusting to their new environment. They're synthesizing enzymes and molecules needed for division, but the population isn't increasing much yet. The length of this phase depends on how different the new conditions are from the previous environment.
• Log (exponential) phase: Cells are dividing at their maximum rate, doubling at regular intervals. This is when generation time is measured. The population increases logarithmically, which is why the growth curve appears as a straight line on a semi-log plot.
• Stationary phase: Growth slows and then stops because nutrients are running out and toxic waste products are building up. The rate of cell division roughly equals the rate of cell death, so the total population stabilizes.
• Death (decline) phase: Cells die faster than new ones are produced. Nutrient depletion and waste accumulation continue to worsen. The population drops, often at a logarithmic rate.

Methods for Cell Count Determination

There are two broad categories of cell counting, and the distinction matters: total cell counts include both living and dead cells, while viable cell counts measure only living cells.

Total cell count methods:

• Direct microscopic count uses a counting chamber (like a Petroff-Hausser chamber or hemocytometer) where you visually count cells in a known volume. It's fast but can't distinguish live cells from dead ones.
• Spectrophotometry measures the turbidity (cloudiness) of a liquid culture. More cells scatter more light, giving a higher absorbance reading. This is quick and non-destructive but requires a standard curve to convert absorbance to cell numbers.

Viable cell count methods:

• Plate count method involves serially diluting a culture, spreading samples on nutrient agar, and counting the colonies that grow. Each colony represents one colony-forming unit (CFU), assumed to have originated from a single viable cell. Only plates with 30–300 colonies are considered statistically reliable.
• Most probable number (MPN) is a statistical method. You inoculate multiple tubes of liquid media with serial dilutions and record which tubes show growth. A probability table then estimates the number of viable cells. This method is especially useful for samples with low cell numbers, like water quality testing.

Binary Fission vs. Other Reproduction

Binary fission is the most common mode of bacterial reproduction. A single parent cell replicates its DNA, elongates, and divides into two genetically identical daughter cells. Because each division doubles the population, growth is exponential under favorable conditions.

Not all microbes reproduce this way:

• Budding: A small outgrowth (bud) forms on the parent cell, enlarges, and eventually separates. The parent and bud are not equal in size. This is characteristic of some yeasts (Saccharomyces cerevisiae) and certain bacteria in the phylum Planctomycetota.
• Fragmentation: Filamentous organisms break into pieces, each capable of growing into a new organism. You'll see this in molds and filamentous bacteria like actinomycetes.
• Spore formation: Some microbes produce specialized reproductive or survival structures. Bacterial endospores (produced by Bacillus and Clostridium) are highly resistant dormant structures that aren't primarily reproductive but allow survival under extreme conditions. Fungal spores, on the other hand, are true reproductive structures produced by molds and mushrooms, and they come in both sexual and asexual varieties.

Biofilms: Formation and Implications

A biofilm is a structured community of microorganisms attached to a surface and encased in a self-produced matrix of extracellular polymeric substances (EPS). The EPS matrix is made of polysaccharides, proteins, and DNA. It protects the community and allows nutrient exchange between cells.

Biofilm formation follows a predictable sequence:

1. Attachment: Free-floating (planktonic) cells reversibly attach to a surface, then irreversibly adhere.
2. Microcolony formation: Attached cells divide and begin producing EPS.
3. Maturation: The biofilm develops complex three-dimensional architecture, including water channels that deliver nutrients and remove waste.
4. Dispersal: Some cells detach from the mature biofilm and return to a planktonic state, colonizing new surfaces.

Biofilm cells can be up to 1,000 times more resistant to antibiotics than planktonic cells of the same species. The EPS matrix physically blocks antimicrobial agents, and cells deep within the biofilm often enter a slow-growing or dormant state that makes them less susceptible to drugs that target active cellular processes.

Biofilms cause significant problems in healthcare and industry:

• Medical device infections: Catheters, prosthetic joints, and heart valves are common sites for biofilm-associated chronic infections.
• Dental plaque: A classic biofilm that leads to tooth decay and periodontal disease.
• Industrial biofouling: Biofilms clog pipes, corrode surfaces, and contaminate water distribution systems.

Quorum Sensing in Microbial Behavior

Quorum sensing is a cell-to-cell communication system that lets bacteria coordinate behavior based on population density. Bacteria release small signaling molecules called autoinducers into their environment. As the population grows, autoinducer concentration rises.

Once autoinducers hit a threshold concentration, they bind to specific receptors on or in bacterial cells, triggering changes in gene expression across the population. Think of it as bacteria "voting": once enough individuals are present, the group acts collectively.

Quorum sensing regulates several important behaviors:

• Bioluminescence in Vibrio fischeri, which lives symbiotically in the light organ of the Hawaiian bobtail squid. Light production only makes sense when enough bacteria are present to generate a visible glow.
• Virulence factor production in pathogens like Pseudomonas aeruginosa. The bacteria wait until their numbers are high enough to overwhelm host defenses before launching a coordinated attack.
• Biofilm formation and dispersal, allowing populations to collectively decide when to establish or abandon a biofilm based on environmental conditions.

Environmental Factors Affecting Microbial Growth

Several environmental factors determine whether microbes can grow and how fast they'll divide:

• Nutrients: All microbes need essential elements like carbon, nitrogen, and phosphorus, plus trace elements (iron, zinc, manganese). Some microbes also require growth factors, which are organic compounds (like vitamins or amino acids) they can't synthesize on their own.
• Temperature: Each species has a minimum, optimum, and maximum growth temperature. Microbes are classified by their preferred range:
  • Psychrophiles thrive in cold (below ~15°C)
  • Mesophiles prefer moderate temperatures (~20–45°C; most human pathogens fall here)
  • Thermophiles grow best at high temperatures (~45–80°C)
• pH: Most bacteria grow best near neutral pH (6.5–7.5), but acidophiles thrive in acidic conditions and alkaliphiles prefer basic environments.
• Oxygen requirements: This is a major way to classify microbes:
  • Obligate aerobes require oxygen
  • Obligate anaerobes are killed by oxygen
  • Facultative anaerobes prefer oxygen but can grow without it
  • Aerotolerant anaerobes don't use oxygen but aren't harmed by it
  • Microaerophiles need oxygen but only at lower-than-atmospheric concentrations

Strategies for Microbial Growth Control

Controlling microbial growth relies on physical, chemical, and environmental approaches.

Physical methods remove or inactivate microbes directly:

• Heat sterilization: Autoclaving (121°C, 15 psi, 15 minutes) kills all microbes including endospores. Incineration destroys contaminated materials completely.
• Filtration: Membrane filters (typically 0.22 µm pore size) physically remove microbes from liquids. HEPA filters do the same for air.
• Radiation: UV light damages microbial DNA by causing thymine dimer formation. Ionizing radiation (gamma rays) penetrates more deeply and is used to sterilize medical supplies and some foods.

Chemical methods use antimicrobial agents:

• Disinfectants are for inanimate surfaces (alcohol, bleach/chlorine, quaternary ammonium compounds).
• Antiseptics are safe for living tissue (iodine, chlorhexidine, hydrogen peroxide).
• Antibiotics target specific microbial processes like cell wall synthesis (penicillin), protein synthesis (tetracycline), or DNA replication (fluoroquinolones).

Environmental control manipulates conditions to prevent growth:

• Temperature control: Refrigeration (slows metabolism) and freezing (halts growth but doesn't necessarily kill).
• pH control: Acidification (pickling, fermentation) or alkalinization creates unfavorable conditions.
• Moisture control: Desiccation and reducing water activity ($a_w$) limit the water available for microbial metabolism. Salting and sugaring foods work on this principle.
• Competitive exclusion: Introducing beneficial microbes (probiotics) that outcompete pathogens for nutrients and attachment sites.