Microbiology Chapter 9: Microbial Growth

Microbial Growth

Increase in number of cells (population)

Biofilms

Colonies of microorganisms (bacteria, fungi, algae) in slimy matrixes on surfaces (ponds and wet rocks) that communicate via cell signaling

What is the most notable characteristic of Biofilms?

Highly resistant to antibiotics and host immune defenses

Form on medical devices, as dental plaque, in cystic fibrosis lungs, and on heart tissue (endocarditis)

Binary Fission

Asexual reproduction - the most common cell replication mechanism in bacteria ; 3 steps

What is the first step of Binary fission?

DNA replication - origin of replication; cell elongates

What is the second step of Binary fission?

Division septum formation = cytokinesis

→ directed by protein FtsZ; forms Z ring (center of cell) → recruits proteins forming divisome that builds the peptidoglycan septum

What is the third step of Binary fission?

Cell separation - two daughter cells; 1 chromosome each

Generation time (doubling time)

Time for a population to double by one round of binary fission

Ex. E. coli: as little as 20 minutes in optimal lab conditions

M. tuberculosis: 15-20 hours; M. leprae: 14 days

Generation growth

Exponential growth → cells = 2^n (n - number of generations)

1 cell dividing every 30 min for 24 h → 48 generations

→ 2^48 ≈ 2.8 × 10^14 cells

Closed (batch) culture

A system where there are no additions (nutrients) or removals (waste products) during the period of incubation | 4 phases

What is the first phase of Closed (batch) culture?

Lag → cell number stagnant; cells create proteins and repair damage

What is the second phase of Closed (batch) culture?

Log (exponential) → binary fission (intrinsic growth rate); susceptible to antibiotics

What is the third phase of Closed (batch) culture?

Stationary - nutrients run out, waste builds → new cells become dying cells (carrying capacity); endospore formation

What is the fourth phase of Closed (batch) culture?

Death - decrease in living cells; persisters survive

Chemostat

Continuous culture (nutrients added, waste removed) → keep cells in log phase

Bacterial counts

Indicate the extent of an infection

→ used for quality control of water, food drugs and cosmetics

Direct Method of Bacterial Counts

Measure cell activity/presence by counting

What are the 3 direct methods for bacterial counts?

Direct microscopic count (Petroff-Hausser chamber) and Coulter counter

Viable plate count (pour plate, spread plate)

Most probable number (MPN)

Petroff-Hausser Chamber

Known volume on a calibrated grid; cells counted under a microscope → easy, fast, inexpensive

Ex: 10 cells in 0.000008 mL → 1,250,000 cells/mL; poor for dilute cultures

Coulter Counter

Each cell passing through small aperture changes electrical resistance → counted (cannot tell live from dead)

Viability (live/dead) stains

Green enters all cells, red only cells with damaged membranes → live = green, dead = red

Viable plate count

Live cells (visible colonies) → reported as CFU/mL (colony-forming units)

Colony might come from multiple cells, possibly some nonculturable cells → low estimate

Count plates with 30-300 colonies (< 30 unreliable; > 300 overcrowded)

Serial Dilution

Multi-step sequence of single dilutions

1.0 mL into 9.0 mL broth, repeated → 1:10, 1:100, 1:1000 ..., then plate each dilution

Ex: 50 colonies from 0.1 mL of the 1:10,000 dilution → 50 × 10 × 10,000 = 5,000,000 CFU/mL

Pour plate method

Method where liquid sample microorganisms are mixed into melted agar (45-50°C) before solidification in sterile dish

Spread plate method

Diluted sample placed onto solid medium and spread on solid agar with sterile spreader

Most Probable Number (MPN)

Statistical estimate of viable cells; growth seen as turbidity or color change

Example: Coliforms (gram-negative lactose fermenters → sign of fecal contamination) in pond water

• 5 lactose broth tubes each get 10 mL, 1 mL, 0.1 mL;

positive = red → yellow

• Result 5-2-0 positive tubes → MPN table → 49 bacteria/100 mL

Indirect Methods of Bacterial Counts

Measure cell presence/activity without counting

What are the 3 indirect methods for bacterial counts?

Turbidity: measured with a spectrophotometer

Dry weight: concentrate, wash, and dry the cells → useful for filamentous microbes

Metabolic activity: ATP formation, protein and nucleic acid synthesis, O2 consumption → fast estimate of live cells

Spectrophotometer

Light passes through the culture; detector measures what passes through

→ More cells → more turbidity → ↑ absorbance (optical density) and ↓ % transmission

Dry Weight Measurement

Cell suspension → concentrated by filtration or centrifugation, washed, dried, and then weighted

Metabolic Activity Measurement

Measurement of cell activity by the production of metabolic products or disappearance of reactants

→ ATP formation, biosynthesis of proteins + nucleic acids, and consumption of oxygen → monitored to estimate # of cells

Fragmenation

Nucleoids that form a filament → multiple new cells split at once

(cyanobacteria, actinomycetes)

Budding

Narrow extension at one pole → swells into smaller bud that detaches

(yeast, prosthecate bacteria, Gemmatta)

Epulopiscium

Daughter cells grow in parent → parent disintegrates to release

Soil Acitinomycetes

Biofilm forms by flow rate

Streamers (fast flow)

Mushroom-like (slow flow)

Composition of Extracellular Polymeric Substances (EPS)

Cells in matrix of EPS (polysaccharides, proteins, nucleic acids) + water channels

→ bring nutrients in, waste out; protect from predation (protozoans, wbc)

Life Cycle of a Biofilm

1) Planktonic cell attaches, becomes sessile (reversible attachment)

2) Irreversible attachment of first colonizers

3) Growth

4) EPS and channels

5) Dispersal

Quorum Sensing

Cells detect density by releasing/sensing small diffusible molecules; autoinducers

Occurs across different microbial species within a biofilm

ex. Candida albicans

Quorum threshold

At threshold (quorum): autoinducer binds receptor → gene expression changes ( sometimes more auto inducers)

Quorum sensing gene activation

Turns on genes that are useful at high density ex. Virulence factors (enough cells to defeat host defense

Gram-negative vs. Gram-positive quorum signals

Gram-negative → N-acylated homoserine lactones

Gram-positive → short peptide

Beneficial Biofilms

Normal microbiota lining intestinal + respiratory mucosa → ward off pathogens

Harmful Biofilms

Dental plaque, wounds, medical devices, (contact lenses, catheters, artificial joints), filtration systems, Pseudomonas aeruginosa (cystic fibrosis)

Biofilm Pathogen Antibiotic Resistance

Deep cells = metabolically inactive (slowing drug uptake) + EPS matrix physically slows the diffusion of antibiotics and antiseptics

EPS matrix protects from host immune cells (wbc)

More efflux pumps out drugs; ideal for resistance genes exchange

Prevention (ISS, 2023): antibacterial coatings fail (dead cells = anchors)

→ surfaces infused with lubricant block microbial adhesion

Great Oxygenation Event

Cyanobacteria released O2 from photosynthesis → O2 rose → mass extinction

Most organisms didn't survive reactive oxygen species (ROS) → damaged macromolecules; Organisms with ability to detoxify ROS → used O2 for energy and lived

Anaerobic environments - sewers, deep ocean, earth's crust

Thioglycolate tube

O2 diffuses in from the top → growth where O2 suits the organism

Anaerobic jar

Chemical packs remove O2 + release CO2

Anaerobic chamber

Enclosed box with all O2 removed; sealed gloves let you handle cultures

Capnophiles

Need more CO2 + less O2 than air → candle jar: the burning candle consumes O2 and releases CO2

Reactive Oxygen Species (ROS)

From aerobic respiration:

superoxide, peroxides, hydroxyl radical, singlet oxygen, hypochlorite

→ must be detoxified

3 detoxifying enzymes

Superoxide dismutase (SOD) - converts dangerous superoxide free radicals into hydrogen peroxide and oxygen

Catalase - degrades hydrogen peroxide into water and oxygen gas

Peroxidase - detoxifies hydrogen peroxide

Obligate anaerobes (ROS enzyme)

Possess no detoxification enzymes, leaving them completely vulnerable to oxygen toxicity.

Aerotolerant anaerobes (ROS enzyme)

Possess Superoxide dismutase (SOD) but lack catalase.

Obligate aerobes (ROS enzyme)

Possess all three detoxification enzymes (peroxidase, SOD, and catalase) to safely survive in oxygen.

Catalase test

Drop of 3% H2O2 → bubbles = catalase positive

(staphylococci +, streptococci −)

Acidity

[H+] measured as pH: below 7 acidic, above 7 basic

Extreme pH damage to macromolecules

High pH → hydrogen bonds in DNA break; extremely basic pH hydrolyzes lipids

Neutralized H+ → proton motive force collapses → impaired ATP production

Proteins = most sensitive: altered ionization and H bonding → misfolding and denaturation

Preservation of acidic foods

Acidic foods resist spoilage - most spoilage microbes prefer near-neutral pH | exception of lactic acid bacteria (thrive near pH 4)

Neutrophiles

Grow near pH 7 (most bacteria), fungi prefer pH 5-6

Acidophiles

Optimum below pH 5.5 → Sulfolobus, Ferroplasma (pH 0-2.9)

→ Adaptations: negatively charged proteins; H+ pumps

Alkaliphiles

Optimum pH 8-10.5 → Natronobacterium, Vibrio cholerae

→ Adaptations: Na+ gradient instead of H+; higher isoelectric point

Psychrophiles

optimum ≈ 15 °C; grow at 0 °C and below; not above ≈ 20 °C

Psychrotrophs

4 °C to 25 °C

Mesophiles

20-45 °C; human pathogens and normal microbiota (37 °C)

Thermophiles

Optimum 50-80 °C

Hyperthermophiles

80-110 °C (some above 121 °C)

Where do Psychrophiles live?

Permanently cold places (deep oceans, polar lakes)

Where do Psychrotrophs live?

Spoil refrigerated food;

Listeria monocytogenes grows at 4-10 °C → risk in pregnancy; avoid soft cheeses, cold cuts

Where do Mesophiles live?

Most human pathogens (E. coli, Salmonella)

Where do Thermophiles live?

Hot springs, compost; Thermus aquaticus, Geobacillus

Where do Hyperthermophiles live?

Deep-sea vents (up to ≈ 340 °C);

Pyrolobus and Pyrodictium grow at 105 °C and survive autoclaving

Osmotic Pressure

High osmotic pressure pulls water out of the cell

→ plasmolysis and death

→ salting preserve food

Halophiles

Require high salt

Halotolerant

Survive in high salt but do not require it

Photosynthetic bacteria

Depend on visible light for energy

Photoautotrophs

Inorganic carbon source, fixes CO2

(cyanobacteria, green sulfur bacteria)

Photoheterotrophs

Organic compounds, cannot fix CO2 (purple nonsulfur bacteria)

Complex media

Yeast, meat, or plant extracts → unknown exact composition (nutrient broth, TSB, brain heart infusion)

Chemically defined media

Every component and amount known (e.g., EZ medium)

Enriched media

Added growth factors, vitamins, and nutrients for fastidious organisms.

Enrichment cultures: favor a microbe present in small numbers

e.g., crude oil as the only carbon source → oil-degrading bacteria

Non-selective media

Support all microbes without inhibition, e.g., Mueller-Hinton agar (Kirby-Bauer antibiotic test)

Selective media

Promote growth of specific target media while suppressing/killing unwanted background microbes

MacConkey agar: bile salts + crystal violet inhibit many Gram-positives → favors Gram-negative enterics

Differential Media

Help distinguish bacteria by the color of the colonies or the change in the medium

MacConkey: lactose fermenters make acid → neutral red turns hot pink