BIOL 2460 chapter 9

Binary Fission is the

Most common form of bacterial reproduction

Binary Fission 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

FtsZ

a protein that forms a ring (Z ring) along the mid-cell division plane to initiate cell division. FtsZ assembles Z ring to for divisome.

Divisome

-a complex of proteins that directs cell division processes in prokaryotes

-activates productionof peptidoglycan and septum

Generation Time (Doubling Time)

time takes to double population

-E. coli = 20 min

-B. subtilis = 120 min

-S. aureus = 30 min

-M. tuberculosis = 15-20 hrs.

Calculating Population Size

Nn=N02n

Nn - number of cells at generation

n - number of generations

N0 - initial number of cells

Lag phase

Inoculum cells added and adjusted to culture medium; no change in population. No increase in the number of living bacterial cells

Log (exponential) phase

Binary fission occurs; cell replication > cell death, exponential increase in the number of living bacterial cells. Most susceptible to disinfectants and antibiotics that affect protein, DNA, and cell wall synthesis.

Stationary phase

Resources become depleted, cell replication = cell death. Plateau in the number of living bacteria cells; the rate of death and division is roughly equal.

Death phase

endospores can form, cell replication < cell death

Chemostat

a device that allows for the continuous culture of microorganisms

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, green is live cells

Coulter counter

-detects electrical resistance change due to cell density

-does not differentiate live/dead

-not good for dense cultures

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)

-countable range is traditionally 30-300 CFU/ml(statistically most accurate)<30 - TFTC >300 - TNTC (too few to count/ too numerous to count)

Membrane filtration technique

-Used to count microbes in samples with very low numbers.

-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

Optical Density (turbidity)

Measured w/ spectrophotometer

•Light is passed thru culture and measured on other side. (More light means less dense and vice versa)

•Population increase = turbidity increase

•Doesn't distinguish live and dead cells.

Biofilm Formation

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

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

Steps of biofilm formation

1. (reversible) Attachment of planktonic cells to a substrate (seconds)

2. Attachment becomes irreversible; cells become sessile (seconds, minutes)

3. Growth & division on substrate (hours, days)

4. Production of extracellular polymeric substance (EPS) (hours, days)

5. Attachment of secondary colonizers & dispersion of microbes to new locations (days, months)

Autoinducer

small molecules are produced to induce various actions

-Gram-negative: N-acylated homoserine lactones

-Gram-positive: short peptides

Quorum Sensing (QS)

cell to cell communication within biofilms

Optimal oxygen concentration

ideal concentration of O2. Organisms will not grow outside of max and min range.

Minimum permissive oxygen concentration

The lowest concentration of oxygen that allows growth

Maximum permissive oxygen concentration

highest O2 concentration allowing growth

Obligate aerobes

require oxygen

-Obligate = must have

-Aerobe = prefers O2

(growth only at the top)

Obligate anaerobes

poisoned by oxygen

-Obligate = must have

-Anaerobe = prefers other than O2

(growth only at the bottom (devoid of O2))

Facultative anaerobes

can live with or without oxygen

-Facultative = can do both

-Anaerobe = prefers other than O2

(growth primarily at the top, but growth distributed evenly throughout the media)

Aerotolerant anaerobes

tolerate but cannot use oxygen

-Aerotolerant = tolerant

-Anaerobe = prefers other than O2

(growth evenly distributed throughout the whole media)

Microaerophiles

require oxygen concentration lower than air (grow at a small distance from the top)

Fluid Thioglycolate Medium (FTM)

low percentage agar tube that has a gradient of oxygen

Acidic

pH less than 7

Basic

pH greater than 7

Optimal growth pH

most favorable pH for growth

Minimum growth pH

lowest pH for growth

Maximum growth pH

highest pH for growth

Neutrophiles

pH ~7

Acidophiles

pH <5.5

Alkaliphiles

pH 8-10.5

Mesophiles

20-45 °C (human pathogens)

Psychrotrophs

4-20 °C (refrigerator)

Psychrophiles

<0 °C (Antarctic, deep freezer)

Thermophiles

50-80 °C

Hyperthermophiles

80-110 °C;some survive @ >121 °C

Halophiles

salt/solute lovers; found in oceans

Halotolerant

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

Photoautotrophs

cyanobacteria and green sulfurs

Photoheterotrophs

purple nonsulfurs

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 the organism of interest

Enrichment cultures

promote growth of the desired organism; it only represents a fraction present

Differential media

distinguish colonies of bacteria by color change

Closed culture

A culture which has no exchange of nutrients or gases with the external environment. Have finite resources (nutrients)

Culture density

the number of cells per unit volume. # of cells/unit vol

Persisters

slowing or stopping of metabolism so that the microbe can't be harmed by the antibiotic

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

Pour plate method steps

1. bacterial sample mixed with warm agar (45-50 c)

2. Sample poured onto sterile plate

3. sample swirled to mix, allowed to solidify

4. plate incubated until bacterial colonies grow

Spread plate method steps

1) Sample (0.1 mL) poured onto solid medium

2) Spread sample evenly over the surface

3) plate incubated until bacterial colonies grow on the surface on the medium

Dilution Factor

the ratio of the initial and final solution volumes

10-1: (1:10)

10-2: (1:100)

10-3: (1:1000)

10-4: (1:10000)

10-5: (1:100000)

extracellular polymeric substance (EPS)

a glycocalyx that helps cells in a biofilm attach to their target environment and to each other. Hydrated polysaccharide gel with other macromolecules and channels.

Biofilms in the body

Normal biota in lungs=good

Plaque formation on teeth bad

Biofilms and antibiotics

May prevent antibiotics and antibodies from reaching bacteria.

1. cells in deep layers may be metabolically inactive

2. EPS may be slow diffusion of biocidal agents

3. Provide optimal environments for sharing of plasmids