UTA Micro 2460 Chapter 9

Binary Fission

Most common form of bacterial reproduction

Binary Fission

4Basic Steps

1.Growth of cell size and increase in cell components

2.Replication of DNA

3.Division of the cytoplasm (cytokinesis)

4.Septum formation and division of daughter cells

Z ring Assembly

•Cytokinesis is directed by FtsZ protein

•FtsZ assembles Z ring to form divisome

•Divisome activates production of peptidoglycan and septum

Generation Time

•Generation Time (Doubling Time) - time takes to double population

Generation Time

among species

•E. coli = 20 min. S. aureus = 30 min.

B. subtilis = 120 min.M. tuberculosis = 15-20 hrs

Calculating Population Size

•Growth is exponential (if resources are no concern)

•Population can be predicted from any starting size

Nn= N02n

Nn - number of cells at generation n

n - number of generations

N0 - initial number of cells

•

•Number of generation may need to be calculated

•Ex. Generation time of 30 min = 48 generations in 24 hours

1 initial cell would result in more than 281 trillion cells after 1 day!

Growth Curve

•Closed cultures have finite resources (i.e. nutrients)

Culture density

the number of cells per unit volume

growth curve phases

lag phase, log phase, stationary phase, death phase

1.Lag phase

- inoculum cells added and adjust to culture medium; no change in population

2.Log

(exponential) phase - binary fission occurs;cell replication > cell death

3.Stationary phase

resources become depleted cell replication = cell death

4.Death phase

1.Death phase - endospores can form cell replication < cell death

Lag Phase

•Initial cell numbers do not change

•Cells grow larger; metabolically active

•Damaged or shocked cells undergo repair

•Duration of the lag phase determined by many factors including:

•Genetic make-up

•Media composition

•Initial inoculum size

Log Phase

•Generation time is genetically determined (intrinsic growth rate)

•Time vs. # of cells is exponential (semilog displays linear)

•Constant growth & uniform metabolism; good for industrial applications

•Most susceptible to disinfectants and antibiotics that affect protein, DNA, and cell-wall synthesis

Intrinsic Growth Rate

Generation time is genetically determined

Stationary Phase

•Waste accumulates; nutrients gradually used up

•Culture density is constant

•Cells enter survival mode; synthesis slows; less susceptible to antibiotics

•Undergo sporulation for endospore-formers

•Expression of virulence factors and secondary metabolites

Death Phase

•Toxic waste accumulates; nutrients exhausted

•Cells lyse and release nutrients for surviving cells and endospore-formers

•Persisters- surviving cells with slow metabolism

•Chronic infections (e.g. tuberculosis); antibiotic resistance

Persisters

Surviving cells with slow metabolism

Sustaining Growth:

•Open system cultures have infinite resources

•Nutrients & air are replenished

•Dead cells & waste are removed

•Beneficial for industrial microbiology

Measuring Growth

•Quantifying populations size is important for determining infection, contamination of water or food supply, etc.

Measuring Growth

Methods

Methods:

•Microscopic cell count

•Fluorescent staining for alive & dead cells

•Coulter count

•Viable cell count

•Optical Density

Direct microscopic cell count

cells are counted under a microscope

•Known volume is transferred to a calibrated slide (Petroff-Hausser chamber) and cells are manually counted

•Cannot distinguish live vs. dead

Fluorescence Staining

•cells are counted under a microscope or flow cytometer

•Red stain binds to damaged cells to indicate dead cells

Coulter counter

•detects electrical resistance change due to cell density

•Does not differentiate live/dead

Viable plate counts

•count of viable cells; samples are diluted and grown on solid media

•Results expressed in colony forming units per volume (CFU/ml)

•Limited only to easily cultured species

•Serial dilution is plated and counted via pour plate or spread plate technique

•Countable range is traditionally 30-300 CFU/ml (statistically most accurate)

<30 - TFTC >300 - TNTC

serial dilution method

•Counting viable CFU requires distinguishable colonies

•Achieved thru serial dilution to achieve the 30-300 CFU/ml range

•Often dilutions are on log scale

•Dilution "factor" is used to determine original CFU count

Dilution Factor

is used to determine original CFU count

pour plate method

A method of inoculating a solid nutrient medium by mixing bacteria in the melted medium and pouring the medium into a Petri dish to solidify

Spread Plate Method

a plate count method in which inoculum is spread over the surface of a solid culture medium

Measuring growth

Dilute Samples

•Very dilute samples (e.g. drinking water) may not contain enough microbes for plate count

•Sample concentrated instead of diluted

•Membrane filtration technique - known vol. filtered through a membrane; membrane plated and colonies counted

Membrane Filter Technique

known vol. filtered through a membrane; membrane plated and colonies counted

Most probable number (MPN)

•- statistical method used when counts are very low (<30 CFU/ml)

•Used in water & food testing

•Uses 3 log dilutions (ex: 1/1, 1/10, 1/100) grown in 3-5 replicates

•Growth is determined positive or negative

•Pattern is compared to reference table (Appendix B)

most probable number method

Optical Density

(turbidity)

•Measured w/ spectrophotometer

•Light is passed thru culture and measured on other side

•Population increase = turbidity increase

Alternate patterns of growth

Some divide asymmetrically (budding) or fragmentation

Fragmentation in

cyanobacteria

Budding of Planctomycetes:

Gemmata obscuriglobus

Biofilm Formation

•Micro ecosystem of one or more species that can provide protection

•Forms mostly in liquid environments (rivers, pipelines, oral cavity)

Biofilm structure:

-clusters of microbes in a matrix

•Extracellular polymeric substances (EPS)

•Secreted by organisms in the biofilm

•Hydrated polysaccharide gel with other macromolecules and channels

Biofilm Formation

Steps of formation:

1.Attachment of planktonic cells to a substrate

2.Attachment becomes irreversible; cells become sessile

3.Growth & division on substrate

4.Production of extracellular polymeric substance (EPS)

5.Attachment of secondary colonizers & dispersion of microbes to new locations

Biofilm Formation

Formed thru:

•Formed through quorum sensing, or cell to cell communication

•TED talk

•Cell density or cellular stress

•Autoinducer small molecules are produced to induce various actions

•Classes: N-acylated homoserine lactones (Gram -) Various short peptides (Gram +)

Autoinducer

a small signal molecule that takes part in quorum sensing

Quorum Sensing molecule classes:

•Classes: N-acylated homoserine lactones (Gram -) Various short peptides (Gram +)

Environmental Factors & Generation Time

•For every extreme, there is likely a species

Main factors that affect growth:

1.Oxygen Level

2.pH

3.Temperature

4.Osmotic pressure

5.Barometric pressure

Oxygen Requirements

•O2 is not always needed or tolerated (Recall: anaerobic respiration)

•Many environments do not have O2

Oxygen Requirements

Levels

•Optimal oxygen concentration -ideal concentration of O2

•Minimum permissive oxygen concentration - lowest O2 concentration allowing growth

•Maximum permissive oxygen concentration - highest O2 concentration allowing growth

•Organism will not grow outside min and max range

Optimal oxygen concentration

ideal concentration of O2

Minimum permissive oxygen concentration

lowest O2 concentration allowing growth

Maximum permissive oxygen concentration

highest O2 concentration allowing growth

Respiration Terminology

•Oxygen requirement can be used to group microbes:

•Obligate aerobes

•Obligate anaerobes

•Facultative anaerobes

•Aerotolerant anaerobes

Microaerophiles

oxygen requirements for Microbial growth

•Obligate = must have

•Facultative = can do both

•Aerotolerant = tolerant

•Aerobe = prefers O2

•Anaerobe = prefers other than O2

Fluid Thioglycolate Medium (FTM)

• - low percentage agar tube that has a gradient of oxygen

Aerotolerance

is determined by location of growth

obligate anaerobes

1.organisms that cannot live where molecular oxygen is present

2.Ex. Bacteroides spp

obligate aerobes

1.require oxygen

2.Ex. Micrococcus luteus

falcultative anaerobes

1.can survive with or without O2

2.Ex. Staphylococcus spp.

aerotolerant anaerobes

tolerate but cannot use oxygen

•Ex. Lactobacillus spp.

•Microaerophiles

can use oxygen only when it is present at levels reduced from that in air 1-10%

•Ex. Campylobacter spp.

Anerobic jars or anaerobic chambers

remove O2

•Studying obligate anaerobes requires special equipment

pH

•pH can affect efficacy of macromolecules; most vulnerable are proteins

•Microbes can prefer acidic (<7) or basic (>7)

•Fermenters are mostly adapted to acidity (think pickles!)

pH Requirements

•Optimal growth pH - most favorable pH for growth

•Minimum growth pH - lowest pH for growth

•Maximum growth pH - highest pH for growth

•Organisms have min. pH, optimal pH, & max. pH

pH requirements for growth

•Groups:

•Neutrophiles pH ~7

•Acidophiles pH <5.5

•Alkaliphiles pH 8-10.5

Temperature

•Microbes also have optimal range for temp.

•Optimal, Min. & Max. temperature

Temperature Requirements

•Mesophiles

= 20-45 °C

•Psychrotrophs

Temperature Requirements

= 4-20 °C

•Psychrophiles

Temperature Requirements

= <0 °C

Thermophiles temp

•Thermophiles = 50-80 °C

Hyperthermophiles Temp

•Hyperthermophiles = 80-110 °C; some survive @ >121 °C

Osmotic Pressure & Growth

•Solute concentrations outside the cell can have effects

•Halophiles - salt/solute lovers; found in oceans

•Halotolerant - tolerate high salt; salt marshes where high solutes aren't present all the time (Recall: MSA & S. aureus)

•

•Halophiles

"salt-loving" archaea that live in environments that have very high salt concentrations

Ex. Reg Alga and Halobacterium

•Halotolerant

tolerate high salt; salt marshes where high solutes aren't present all the time (Recall: MSA & S. aureus)

ex. Halomonas spp.

isotonic solution

Osmotic Growth

NO net movement of water particles, cell membrane is attached to cell wall

hypertonic solution

Osmotic Growth

Water particles move out of the cell, cell membrane shirks and detaches from cell wall. Plasmolysis

hypotonic solution

Osmotic Growth

Water particles move into cell, cell wall counter acts osmotic pressure. To prevent swelling and lysis

Media Used for Bacterial Growth

•All-purpose media (e.g. TSA)

•Enriched media - contains growth factors, vitamins, and other essentials to promote growth of :

•Fastidious organisms- cannot make certain nutrients

•Chemically defined medium- complete chemical composition known

•Complex medium- contains extracts and digests of yeasts, meat, or plants; exact composition not known

•Selective media - inhibit unwanted, promote growth of organism of interest

•Enrichment cultures promote growth of desired organism; only represents a fraction present

•Differential media - distinguish colonies of bacteria by color change

•Enriched media

-contains growth factors, vitamins, and other essentials to promote growth of :

•Fastidious organisms- cannot make certain nutrients

Chemically defined medium-

- complete chemical composition known

Complex medium

- contains extracts and digests of yeasts, meat, or plants; exact composition not known

Selective media

- inhibit unwanted, promote growth of organism of interest

Enrichment cultures

promote growth of desired organism; only represents a fraction present

Differential media

- distinguish colonies of bacteria by color change

All purpose media

supports growth of most microorganisms (nutrient agar & broth, trypticase soy broth) (TSA)

•Barophiles

•Ability to withstand great pressure

- require high atmospheric pressure

•Found on bottom of ocean

•Largely unculturable; not much known

•Also can be thermo or hyperthermophiles

•Photoautotrophs

- cyanobacteria and green sulfurs

Photoheterotrophs

•- purple nonsulfurs

Photosynthetically active radiation (PAR)

The portion of electromagnetic spectrum that is absorbed by organisms

•Usu. within visible light spectrum (400-700nm)

•Accessory pigments :

such as fucoxanthin in brown alga, and phycobilin in cyanobacteria, widen ranges for photosynthesis.

Z-ring function in binary fission

It forms a contractile ring at the septum