Microbio: Module 5: Bacterial & Archael Growth

7.1 Most Bacteria and Archaea Reproduce by Binary Fission

Reproductive Strategies of Bacteria and Archaea

• Most bacterial and archaeal cells reproduce by binary fission.

• Some bacteria reproduce by forming a bud or multiple fission.

• All must replicate and segregate the genome prior to division

t/f bacteria only reproduce by method of binary fission

false

7.2 Bacterial Cell Cycles Are Divided into Three Phases

Bacterial Cell Cycle

The cell cycle is the complete sequence of events extending

from formation of a new cell through the next cell division.

Three phases:

• Period of growth after the cell is born.

• Chromosome replication and partitioning.

• Cytokinesis, during which a septum and daughter cells are

formed

7.2 Bacterial Cell Cycles Are Divided into Three Phases

Chromosome Replication and Partitioning

• Most bacterial have a single circular chromosome.

• Single origin of replication—site at which replication begins.

• Terminus—site at which replication is terminated, located opposite of the origin.

• Replisome—DNA synthesis machinery.

• DNA replication proceeds in both directions from the

origin.

• Origins move to opposite ends of the cell, and the rest of

each chromosome follows.

7.2 Bacterial Cell Cycles Are Divided into Three Phases

Cell Cycle of E. coli

7.2 Bacterial Cell Cycles Are Divided into Three Phases

Partitioning System

Three components:

• ParA protein

• ParB protein

• parS region on chromosome

7.2 Bacterial Cell Cycles Are Divided into Three Phases

Chromosome Partitioning—An Example

C. crescentus

• parS sites are close together near origin of replication.

  • Being early in duplication, it directs the segregation of the two daughter chromosomes.

• ParB proteins bind the parS site and nearby.

• One partition complex remains at the stalk pole, whereas other is guided by ParA to opposite pole.

  • Similar to a baton being passed in relay.

7.2 Bacterial Cell Cycles Are Divided into Three Phases

Cytokinesis and Septation

Cytokinesis—formation of two daughter cells following cell

division.

Septation—formation of cross wall between two daughter

cells.

Septation steps:

• Selection of site for septum formation.

• Assembly of Z-ring (composed of protein FtsZ).

• Assembly of cell wall-synthesizing machinery.

• Constriction of cell and septum formation.

7.2 Bacterial Cell Cycles Are Divided into Three Phases

Z-Ring Formation

FtsZ protein

• Involved in assembly of Z-ring.

• Early, FtsZ filaments are heterogeneous in size and widely distributed around the plasma membrane.

• Min system keeps oscillating between left and right pole without allowing FtsZ at the poles.

• First protein to localize to the future division site.

• FtsZ monomers polymerize.

• Localize with Min system to establish division site at midcell.

7.2 Bacterial Cell Cycles Are Divided into Three Phases

Importance of Z-Ring Timing

Location of FtsZ polymerization must coordinate with timing.

• Too early, Z-ring could constrict preventing proper partitioning.

Nucleoid occlusion

• Coordinates chromosome movement and cell separation.

• SlmA coats chromosome except at the replication termination region.

  • Z-ring can form when SlmA-tagged chromosome has moved away.

7.2 Bacterial Cell Cycles Are Divided into Three Phases

Nucleoid occlusion

• Nucleoid occlusion is a crucial bacterial defense mechanism that stops cell division (cytokinesis) fr happening over the chromosome (nucleoid), preventing its breakage and ensuring proper cell division.

• It works through DNA-binding proteins like Noc or SlmA that bind to specific sites on the DNA, preventing the formation of Z-ring (the division machinery) in that area.

• This ensures the septum forms at the correct mid-cell location, coordinating cell division with chromosome replication and segregation for faithful inheritance of the genome.

How it works:

Inhibition of Z-ring assembly:

• Proteins like SlmA (in E. coli) or Noc (in B. subtilis) bind to specific DNA sequences on nucleoid.

Physical blocking:

• These bound proteins physically interfere with the assembly of FtsZ, the key protein that forms the Z-ring, preventing it from forming over the DNA.

Temporal and spatial regulation:

• By marking the nucleoid, these proteins act as spatial and temporal regulators, ensuring the septum forms away from the DNA, often at mid-cell once replication is complete.

Coordination with replication:

This mechanism links cell division to the cell cycle, preventing division until the chromosome is properly replicated and segregated, thus avoiding catastrophic chromosome breakage

7.2 Bacterial Cell Cycles Are Divided into Three Phases

Divisome Formation

• Divisome formation is a two-stage process in bacteria (in E. coli) where a protein complex assembles at the mid-cell to mediate cell division (septation).

• It begins with the formation of the Z-ring (FtsZ) at the membrane, followed by the recruitment of division proteins (FtsA, ZipA, FtsQLB, FtsW, FtsI, FtsN) that synthesize the septum.

Key Stages and Compoents of Divisome Formation:

Stage 1: Z-ring Assembly: The tubulin-like protein FtsZ polymerizes into a ring structure (Z-ring) at the future site of division, tethered to the inner membrane by FtsA and ZipA.

Stage 2: Divisome Maturation & Recruitment: Early proteins (FtsZ, FtsA, ZipA) recruit late proteins, including the FtsQLB complex, FtsW (transglycosylase), FtsI (transpeptidase), and finally FtsN. FtsN triggers the activation of septal peptidoglycan synthesis.

Function: The divisome acts as a "machine" that synthesizes new cell

wall and membrane material, constricting the cell into two daughter cells

7.2 Bacterial Cell Cycles Are Divided into Three Phases

Peptidoglycan Synthesis Critical in Determination of Shape

Peptidoglycan synthesis scheme:

• NAG-NAM-pentapeptide synthesized.

• MurJ flips the carrier-bound building block across membrane.

• NAG-NAM inserted into peptidoglycan strand.

7.2 Bacterial Cell Cycles Are Divided into Three Phases

Cell Shape Determination

Cellular location of peptidoglycan synthesis plays role in determining cell shape.

Coccus

• Peptidoglycan only forms at central septum.

  • FtsZ localization placement involved.

7.2 Bacterial Cell Cycles Are Divided into Three Phases

Rod and Curved Cell Shape

Rod

• Elongasome (rod complex)

  • MreB is scaffold, by creating filaments along cytoplasmic face of plasma membrane.

  • Growth occurs in numerous bands around cell, not at the poles.

Curved

• Crescentin localizes to one side of the cell, resulting in asymmetric cell wall and vibroid shape

7.3 Archaeal Cell Cycles Are Unique

Archaeal Cell Cycles Resemble Eukaryal Cell Cycles –Except in Segregation of Chromosomes

Studies in Sulfolobus spp. indicate a cycle similar to a mitotic cycle.

• Growth phase (G1) followed by DNA replication (S phase), then G2 phase, segregation of chromosomes, and cytokinesis.

• Segregation occurs via use of SegA/SegB protein system that is similar to bacterial partitioning systems.

  • SegA similar in structure and function to ParA.

  • SegB is unique to archaea, but thought to function similarly to ParB.

• DNA sequences similar to parS have yet to be found in archaea.

7.3 Archaeal Cell Cycles Are Unique

Archaeal Cytokinesis Similar to Eukaryotes

• CdvA—Bind the membrane and forms a non-contractile

ring at midcell.

• CdvB—Ring constricts to separate the daughter cells.

• CdvC—Recruited with CdvB to the site of division.

• FtsZ—Z-ring associates with the new S-layer

7.4 Growth Curves Consist of Five Phases

Growth

Increase in cellular constituents that may result in:

• Increase in cell number.

• Increase in cell size.

Growth most commonly referred to as population growth rather than growth of individual cells

7.4 Growth Curves Consist of Five Phases

Microbial Growth Curve

Observed when microorganisms are cultivated in liquid (broth) culture.

• Batch culture—incubated in a closed vessel with a single batch of medium.

Usually plotted as logarithm of cell number versus time

7.4 Growth Curves Consist of Five Phases

Five Phases of Growth

1. Lag Phase

Cell synthesizing new components.

• Replenish spent materials (that is, ribosomes and ATP).

• To adapt to new medium or other conditions.

• Eventually, cells replicate their DNA, increase in mass, and divide.

2. Exponential Phase

Rate of growth and division is constant and maximal.

Population is most uniform in terms of chemical and physical

properties during this phase.

7.4 Growth Curves Consist of Five Phases

Exponential Phase Growth Rate Depends on Nutrient Availability

During exponential phase, cells grow as quickly as they can for the conditions available.

• Final net growth increases with the initial amount of the limiting nutrient present.

• Growth rate increases with nutrient concentration, but it saturates.

7.4 Growth Curves Consist of Five Phases

Phases of Growth—Stationary Phase

3. Stationary Phase

In a closed system, growth eventually ceases.

Total number of viable cells remains constant.

• Balance between cell division and cell death.

• Population may cease to divide but remain metabolically active.

Reasons for stationary phase:

• Nutrient limitation

• Limited oxygen availability

• Toxic waste accumulation

• Critical population density reached

7.4 Growth Curves Consist of Five Phases

Critical population density

Critical population density" refers to a threshold level in ecology, microbiology, and

behavioral science where a population's density triggers a significant, often rapid,

change in behavior or state. This threshold is a fundamental mechanism for moving from

disorganized, individual actions to highly synchronized group, swarm, or herd behavior.

•Ecological and Microbiological Impact:

• Bacterial Quorum Sensing: Bacteria use this density to switch on group behaviors,

such as biofilm formation, which can be critical for survival.

• Predator-Prey Interactions: The rapid propagation of this density can cause abrupt,

widespread shifts in the balance between predators and prey.

• Antibiotic Resistance: Bacterial populations that reach a certain critical density can

withstand otherwise effective antibiotic treatments.

7.4 Growth Curves Consist of Five Phases

Phases of Growth

4. Death Phase

Number of viable cells declines exponentially, with cells dying

at a constant rate.

Nutrient deprivation and the buildup of toxic wastes cause

irreparable harm to the cells.

5. Long-Term Stationary Phase

Bacterial population continually evolves.

Process marked by successive waves of genetically distinct

variants.

Natural selection occurs within a single culture.

7.4 Growth Curves Consist of Five Phases

Mathematics of Growth

Generation (doubling) time

• Time required for the population to double in size.

• Varies depending on species of microorganism and environment.

7.4 Growth Curves Consist of Five Phases

Calculation of Growth Rate

Calculation of the growth rate constant (k)—number of

generations per unit time.

• Let N0 = the initial population number

• Nt = the population at time t

• n = the number of generations in time t

For populations reproducing by binary fission:

• !

" #!" "= !

Solving for n, the number of generations, where all logarithms are

to the base 10,

• log Nt = log N0 + n * log 2

• n = (log Nt − log N0)/log 2 = (log Nt − log N0)/0.301

Overall:

k = (n/t) = (log Nt − log N0)/0.301t

7.4 Growth Curves Consist of Five Phases

Calculation of Generation Time

Calculation of generation (doubling) time:

If a population doubles, then

• Nt = 2 N0

Substitute 2N0 into the growth rate

constant equation and solve for

• k = (log (2N0) − log N0)/0.301g =

(log 2 + log N0 − log N0)/0.301g

• k = 1/g

The generation time is the reciprocal

of the growth rate constant.

• g = 1/k

7.5 Environmental Factors Affect Microbial Growth

The Influence of Environmental Factors on Growth

• Most organisms grow in fairly moderate environmental conditions.

• Extremophiles—Grow under harsh conditions that would killmost other organisms.

• All microbes must respond to changes in their environment.

• Microbes have an optimal range of an environmental parameter for best growth.

7.5 Environmental Factors Affect Microbial Growth

Solutes Affect Osmosis and Water Activity

Changes in osmotic concentrations in the environment may

affect microbial cells.

Hypotonic solution

• Lower solute concentration outside the cell than inside the

cell.

Water enters the cell and may burst.

Hypertonic solution

• Higher solute concentration outside the cell than inside the cell.

  • Water leaves the cell and membrane shrinks

7.5 Environmental Factors Affect Microbial Growth

Microbes Adapt to Changes in Osmotic Concentrations

Most microbes live in hypotonic environment.

• Protected by cell wall, preventing overexpansion of

plasma membrane.

Mechanisms to lower solute concentration in cytoplasm:

• Mechanosensitive (MS) channels in plasma membrane

allow solutes to leave.

• Protists use contractile vacuoles to expel excess water

7.5 Environmental Factors Affect Microbial Growth

Osmophiles

Halophiles

• Require NaCl at a concentration above about 0.2 M.

• Extreme halophiles

  • Require salt concentrations between 3 M and 6.2 M.

• Salt-in

  • Accumulate K and Cl in cytoplasm.

  • Proteins need these high salt levels.

• Salt-out

  • Keep salt ions outside of cell.

  • Synthesize compatible solutes that do not interfere with growth.

7.5 Environmental Factors Affect Microbial Growth

Solutes and Water Activity

Water activity (aw)

• Measure of the degree of water availability.

  • Low water activity means most water is bound, not available tomicroorganisms.

• 1/100 the relative humidity of solution.

• Equal to ratio of solution’s vapor pressure (Psoln) to that of pure water (Pwater).

• Most microorganisms only grow well at water activities around 0.98.

Osmotolerant—microorganisms that can grow over wide ranges of water activity but optimally at higher levels.

Xerotolerant—microbes that withstand high solute concentrations.

7.5 Environmental Factors Affect Microbial Growth

pH

pH is a measure of the relative acidity of a solution.

• Negative logarithm of the hydrogen ion concentration.

Acidophiles

• Growth best between pH 0 and 5.5.

Alkaliphiles (alkalophiles)

• Growth best between pH 8 and 11.5.

7.5 Environmental Factors Affect Microbial Growth

pH Preference of Microorganisms

Most bacteria and protists are neutrophils.

Most fungi prefer more acidic surroundings, about pH 4 to 6.

• Photosynthetic protists also seem to favor slight acidity.

Some archaea are acidophiles.

Alkaliphiles are distributed among all three domains of life (Bacteria, Archaea and Eukarya).

7.5 Environmental Factors Affect Microbial Growth

pH Tolerance Mechanisms of Microbes

Microorganisms respond to external pH changes using

mechanisms that maintain a neutral cytoplasmic pH.

• Neutrophiles exchange potassium for protons

Acidophiles

• Pump protons (H+) out of the cell.

Alkaliphiles

• Exchange internal !"+ ions for external protons

7.5 Environmental Factors Affect Microbial Growth

Temperature

Microbes cannot regulate their internal temperature.

Enzymes have optimal temperature at which they function

optimally.

Below optimum temperature, enzyme is not catalytic.

High temperatures may inhibit enzyme functioning and be lethal.

Cardinal temperatures

• Minimum

• Maximum

• Optimal

7.5 Environmental Factors Affect Microbial Growth

Temperature Ranges for Microbial Growth

• Psychrophiles—0degC to 20degC

• Psychrotrophs—0degC to 35degC

• Mesophiles—20degC to 45degC

• Thermophiles—45degC to 85degC

• Hyperthermophiles—85degC to 100degC

7.5 Environmental Factors Affect Microbial Growth

Adaptations of Thermophiles

Heat-stable enzymes and protein synthesis systems that

function at high temperatures.

Protein structure stabilized by a variety of means.

• More H bonds.

• More proline, less flexible peptides.

• Chaperones aid in folding.

Membrane stabilized by variety of means.

• More saturated, more branched and higher molecular weight.

• Ether linkages, resistant to hydrolysis.

7.5 Environmental Factors Affect Microbial Growth

Oxygen Concentration

Growth in presence of different oxygen concentrations

depends on a microbe’s metabolic processes, electron

transport chains (ETC), terminal electron acceptor used.

Five types of relationships to O2

• Obligate aerobe—requires O2.

• Obligate anaerobe—usually killed in presence of O2.

• Microaerophile—requires 2 to 10% O2.

• Facultative anaerobes—do not require O2 but grow better in its presence.

• Aerotolerant anaerobes—grow with or without O2

7.5 Environmental Factors Affect Microbial Growth

Basis of Different Oxygen Sensitivities

Oxygen reduced to reactive oxygen species (ROS).

• Superoxide radical

• Hydrogen peroxide

• Hydroxyl radical

Microorganisms must be able to protect itself from ROS.

• Aerobes produce protective enzymes

  • Superoxide dismutase (SOD)

  • Catalase

  • Peroxidase

7.5 Environmental Factors Affect Microbial Growth

Pressure

Microbes that live on land and water surface live at a

pressure of 1 atmosphere (atm).

Some Bacteria and Archaea live in deep sea with very high

hydrostatic pressures.

Barotolerant—adversely affected by increased pressure, but

not as severely as nontolerant organisms.

Peizophilic (barophilic)—requires high pressure for growth.

• Change membrane fatty acids to adapt to increasing pressure.

  • Lipids become more unsaturated and shorter

7.5 Environmental Factors Affect Microbial Growth

Harmful Radiation

Ionizing radiation

• X-rays and gamma rays

• Cause mutations that indirectly result in death.

• Disrupts chemical structure of many molecules.

  • Breaks H-bonds and destroys ring structures.

• Bacterial endospores and Deinococcus radiodurans are extremely resistant to ionizing radiation.

7.5 Environmental Factors Affect Microbial Growth

Radiation Damage—UV and Visible Light

Ultraviolet (UV) radiation

• Most lethal wavelength is 260 nm because it is absorbed

by DNA.

• DNA damage can be repaired by several repair

mechanisms.

Visible light

• At high intensities generates singlet oxygen (¹O₂), a powerful oxidizing agent.

• Carotenoid pigments protect many light-exposed microorganisms from photooxidation

7.6 Microbial Growth in Natural Environments

Microbial environments:

• Are complex and constantly changing.

• Expose a microorganism to overlapping gradients of nutrients and environmental factors.

• Contain both micro- and macroorganisms.

7.6 Microbial Growth in Natural Environments

Most Microbes Live in Growth-Arrested States

Few microbes live in nutrient rich (eutrophic) environments.

Most microbes inhabit low nutrient (oligotrophic)

environments.

Microbes have evolved many responses to starvation and

environmental stress:

• Morphological changes (that is, endospore formation)

• Growth arrest—enter stationary phase.

• Can use cellular components as nutrients.

• Numerous proteins that help.

• Viable but not culturable state—resume growth once

nutrients return.

• Persisters

7.6 Microbial Growth in Natural Environments

Biofilms Are Common in Nature

lots of questions here

• Most microbes grow attached to surfaces (sessile) rather

than free floating (planktonic).

• These complex, slime enclosed communities of microbes

are called a biofilm.

• Biofilms are ubiquitous in nature in water.

• Can be formed on any conditioned surface.

7.6 Microbial Growth in Natural Environments

Biofilm Formation

lots of questions here

• Microbes reversibly attach to conditioned surface and release polysaccharides, proteins, and DNA to form the extracellular polymeric substance (EPS).

• Additional polymers are produced as microbes reproduce

and biofilm matures.

7.6 Microbial Growth in Natural Environments

Heterogeneity in Biofilms

A mature biofilm is a complex, dynamic community of microorganisms.

Heterogeneity is differences in metabolic activity and locations of microbes.

Interactions occur among the attached organisms.

• Use molecules to communicate and DNA uptake can occur in community

7.6 Microbial Growth in Natural Environments

Biofilm Microorganisms

• Emergent properties

• Properties could not be predicted from studying single cell.

• Physiological changes and EPS protect microbes from

harmful agent (that is, UV).

• When formed on medical devices, such as implants,

antibiotic treatment fails.

• Chunks of biofilm can be sloughed off which can

contaminate a drinking water system

7.6 Microbial Growth in Natural Environments

Cell-Cell Communication Within Microbial Populations

Quorum sensing

Bacterial cells communicate via small molecules that diffuse

in the environment.

• In order to conduct business, a sufficient number of

microbes must be present and participating

7.6 Microbial Growth in Natural Environments

Quorum Sensing Systems

N-acylhomoserine lactone (AHL)

• Autoinducer

• Normally, moves across the plasma membrane from the cytoplasm to the outside of the cell.

• When cell population is high, AHL diffuses in the cell which induces specific gene expression.

  • Pseudomonas aeruginosa uses this to express virulence factors.

  • Bioluminescent bacteria use this to light.

Autoinducing peptides (AIPs)

• Gram positive bacteria

• Autoinducing short peptides

7.7 Laboratory Culture of Microbes Requires Conditions that Mimic Their Normal Habitats

Culture Media

Culture medium—solid or liquid mixture of nutrients andother compounds.

• Need to grow, transport, and store microorganisms in lab.

Must contain all growth factors microorganism requires.

Use knowledge about microbe's normal habitat when

selecting culture media.

Classified based on chemical composition, physical nature

and their function

7.7 Laboratory Culture of Microbes Requires Conditions that Mimic Their Normal Habitats

Types of Media

Defined/synthetic medium

• Each ingredient can be defined with a chemical formula.

Complex media

• Contain some ingredients of nonspecific chemical

composition.

7.7 Laboratory Culture of Microbes Requires Conditions that Mimic Their Normal Habitats

Some Media Components

Peptones—partial proteolytic digestion of protein sources.

Extracts—aqueous extracts that contain amino acids,

peptides, nucleotides, organic acids, vitamins, and minerals.

• Usually beef or yeast.

Agar—sulfated polymer solidifying agent.

• Most microorganisms cannot degrade it

7.7 Laboratory Culture of Microbes Requires Conditions that Mimic Their Normal Habitats

Functional Types of Media

Supportive media (that is, tryptic soy broth and agar)

• Sustain growth of many microorganisms.

Enriched media (that is,

blood agar)

• Supportive media supplemented with special nutrients.

7.7 Laboratory Culture of Microbes Requires Conditions that Mimic Their Normal Habitats

Selective and Differential Media

Selective media—allow the growth of particular microorganisms,

while inhibiting the growth of others.

• Gram-negative bacteria can grow on bile salts, while Gram-

positive bacteria cannot.

Differential media—distinguish among different groups of

microbes and even permit tentative identification of microbes

based on their biological characteristics.

• Blood agar—distinguish

between hemolytic versus

nonhemolytic bacteria.

• MacConkey agar—distinguish

between lactose fermenters

versus nonfermenters.

7.7 Laboratory Culture of Microbes Requires Conditions that Mimic Their Normal Habitats

Cultivation of Anaerobic Microbes

These microbes cannot tolerate O2 and must be grown

without O2.

• Anaerobic media with reducing agents

• Anaerobic chamber

• Hard transparent containers sealed tightly.

• Candle Jar

7.7 Laboratory Culture of Microbes Requires Conditions that Mimic Their Normal Habitats

Isolation of Pure Cultures

• Pure or axenic culture—population of cells arising from a

single cell.

• Allows for the study of single type of microorganism in

mixed culture.

• To isolate a single bacteria, microbiologists enrich for the

microbe of interest and then follow with methods to obtain

a pure culture

7.7 Laboratory Culture of Microbes Requires Conditions that Mimic Their Normal Habitats

Enrichment Cultures

Enrichment culture

• Encourages growth of microbes with a particular

characteristic, while inhibiting growth of others.

Three factors considered:

• A suitable source of microbes.

• Nutrients that should and should not be included in the

culture medium.

• Environmental conditions

7.7 Laboratory Culture of Microbes Requires Conditions that Mimic Their Normal Habitats

Streak Plate

Technique of spreading a mixture of cells on an agar surface

using an inoculating loop or swab.

• Goal is to obtain individual cells that are separated from

each other.

Each cell can reproduce to form a separate colony (visible

cluster of microorganisms).

7.7 Laboratory Culture of Microbes Requires Conditions that Mimic Their Normal Habitats

Spread Plate

Small volume of diluted mixture is transferred to the center of

an agar plate and spread evenly over surface with a sterile

bent rod.

• Dilution made by serial dilution

7.7 Laboratory Culture of Microbes Requires Conditions that Mimic Their Normal Habitats

Pour Plate

• Sample is serially diluted and mixed with liquid agar.

• This mixture of cells and agar is poured into sterile culture dishes.

• Useful when sampling a heterogeneous population of microbes that might produce overgrown colonies.

7.7 Laboratory Culture of Microbes Requires Conditions that Mimic Their Normal Habitats

New Approaches to Culturing Microbes

Culturomics

• Use miniature cultures and incubate in many different conditions to find the best one.

Bring natural environment into the lab (that is, seawater).

• Diffusion chamber that slows nutrients to diffuse but retains the microbes.

  • Dilution can contain a single cell.

Co-culturing

• Growth on animals or tissues in lab.

7.8 Microbial Population Size Can Be Measured Directly or Indirectly

Measurement of Microbial Growth and Direct Measurement of Cell Numbers

Microbial growth can be measured by changes in number of

cells or mass.

Direct measurement of cell Numbers

• Direct counts

  • Counting chambers—special slides and cover slips with grids to facilitate counting.

  • Membrane filter technique—microbes are filtered and then stained.

  • Flow cytometry—stream of cells so narrow that one cell at a time passes through the laser beam.

  • Electronic counters—the Coulter counter

7.8 Microbial Population Size Can Be Measured Directly or Indirectly

Counting Chambers

Number of microbes calculated from chamber’s volume and dilutions.

Advantages

• Easy, inexpensive, and relatively quick.

• Gives information about the size and morphology of microorganisms.

Disadvantages

• Population must be large and

evenly dispersed.

7.8 Microbial Population Size Can Be Measured Directly or Indirectly

Direct Counts on Membrane Filters

Cells filtered through special membrane that provides dark

background for observing cells.

Cells are stained with fluorescent dyes.

• DAPI

With certain dyes, can distinguish living from dead cells

7.8 Microbial Population Size Can Be Measured Directly or Indirectly

Flow Cytometry

Microbial suspension forced through small orifice with a laser

light beam.

Scattered light detected by flow cytometer.

• Each light scattering event is detected independently, thus the number of events represents the number of cells.

Cells of differing size, internal complexity, and other characteristics within a population can also be counted.

7.8 Microbial Population Size Can Be Measured Directly or Indirectly

Electronic Counters—Coulter Counter

• Microbial suspension is forced through a small hole.

• Electrical current flows through the hole, and electrodes

placed on both sides of the hole measure electrical

resistance.

• Every time a microbial cell passes through the hole,

electrical resistance increases (that is the conductivity

drops), and the cell is counted

7.8 Microbial Population Size Can Be Measured Directly or Indirectly

Viable Counting Method—Standard Plate Counts

Spread and pour plate techniques

• Diluted sample of bacteria is spread over solid agar

surface or mixed with agar and poured into Petri plate.

• After incubation, the numbers of organisms are

determined by counting the number of colonies multiplied

by the dilution factor.

• Results expressed as colony forming units (CFU)

7.8 Microbial Population Size Can Be Measured Directly or Indirectly

Viable Counting Method—Membrane Filter Method

Membrane filter technique

• Bacteria from aquatic samples are trapped on

membranes.

• Membrane soaked in culture media.

• Colonies grow on membrane.

• Colony count determines number of bacteria in sample

7.8 Microbial Population Size Can Be Measured Directly or Indirectly

Measurement of Cell Mass

Dry weight

• Time consuming and not very sensitive.

• Cells from liquid culture are pelleted by centrifuged and weighed.

Spectrophotometry

• Amount of light scattering is directly

proportional to cell biomass.

Concentration of a particular cell constituent

• Concentration of protein or nitrogen

proportional to number of cells

7.9 Chemostats and Turbidostats are Used for Continuous Culture of Microorganisms

Continuous Culture of Microorganisms

Continuous culture system

Maintains cells in exponential growth at a known rate for

extended periods.

Advantages

• Allow study of microbial growth at very low nutrient

concentrations, close to those present in natural

environment.

• Very useful in microbial ecology

7.9 Chemostats and Turbidostats are Used for Continuous Culture of Microorganisms

Chemostats and Turbidostats

• Chemostat

• Rate of incoming medium = rate of

removal of medium from vessel.

• An essential nutrient is in limiting

quantities.

• Most stable and effective at lower

dilution rates.

• Turbidostat

• Regulates flow rate of media through

vessel to maintain predetermined

turbidity.

• Dilution rate varies.

• Contains all nutrients in excess.

• Operates best at high dilution rates

7.9 Chemostats and Turbidostats are Used for Continuous Culture of Microorganisms

Dilution Rate and Microbial Growth

• Dilution rate—rate at

which medium flows

through vessel.

• Note: cell density

maintained at wide

range of dilution rates.

• Population size and

generation time are

related to the dilution

rate.