Micro Ch 9

UTA Dylan Parks

What are the steps of bacterial reproduction?

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 daugther cells

What does the FtsZ protein direct?

Cytokinesis

What forms the Z ring?

FtsZ forms the Z ring to form a divisome

What does the divisome in the Z ring activate?

Activates production of peptidoglycan and septum

What does generation time (doubling time) mean?

Time takes to double population

How long does it take E. coli to double?

20 minutes

How long does it take B. subtilitis to double?

120 minutes

How long does it take S. aureus to double?

30 minutes

How long does it take M. tuberculosis to double?

15-20 hours

Is growth exponential?

Yes, if recources are no concern

Can populations be created from any starting size?

Yes

What is the formula for calculating population size?

N₂=N₀2ⁿ

• n= number of generations

• N₀= initial number of cells

What are the steps of the growth curve?

1. Lag phase

2. Log (exponential phase)

3. Stationary phase

4. Death phase

What happens in the log phase?

Cells adjust to culture medium; no change in population

What happens in the log (exponential phase)?

Binary fission occurs; cell replication > cell death

What happens in the stationary phase?

Resources become depleted; endospores can start forming

Cell replication = cell death

What happens in the death phase?

Endospores persis cell replication < cell death

What are the benefits of measuring growth?

• Open system cultures have inifinite resources

• Nutrients and air are replenished

• Dead cells and waste are removed (effluent)

• Benificial for industrial microbiology

Why is measuring growth important?

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

What are some methods of measuring growth?

• Microscopic cell count

• Fluoresecent staining for alive and dead cells

• Coulter count

• Viable cell count

• Optical density

What is microscopic cell count?

Cells are counted under a microscope

• Known volume is transferred to a calibrated slide and cells are manually counted

• Cannot distinguish live vs. dead

What is coulter counter?

Detects electrical resistance change due to cell density

• Does not differentiate live/dead

What is optical density (turbidity) ?

Measured w/ spectrophotometer (light is passed thru culture and measured on other side.

• Population increase = turbidity increase

• Includes dead and live cells

What is fluorescence staining?

Cells are counted under a microscope or flow cytometer

• Red stain binds to damaged cells to indicate dead cells

What is viable cell count?

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 techique

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

Why are counting CFU and serial dilution important for measuring growth?

• Counting viable CFU requires distinguishable and separate 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

What are the steps to the pour plate method?

1. Bacterial sample mixed with warm agar (45-5*C)

2. Sample poured onto sterile plate

3. Sample swirled to mix, allowed to solidify

4. Plate incubated until bacterial colonies grow

What are the steps to the spread plate method?

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 of the medium

What does the most probable number (MPN) mean?

Statistical method used when counts are very low (<30 CFU/mL)

How is measuring growth used and how?

• Used in water and food testing

• Uses 3 log dilutions (e: 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)

What are some alternate patterns of growth?

Some divide asymmetrically (budding) or fragmentation

What is biofilm formation?

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

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

What are the steps of biofilm formation?

1. Attachment of planktonic cells to as substrate

2. Atttachment becomes irreversible; cells become sessile

3. Growth and division on substrate

4. Production of extracelular polymeric substance (EPS)

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

How is biofilm formed?

Formed through quorum sensing, or cell to cell communication

• Cell density or cellular stress

• Autoinducer small molecules are produced to induce various actions

What are the classes of biofilm?

• N- acylated homoserine lactones (Gram -)

• Various short pepetides (Gram +)

Is biofilm beneficial?

Some are benificial, some are not

Is normal biota in the gut beneficial?

Good

Is plaque formation on teeth beneficial?

Bad

How do biofilms react to antibiotics?

Often provide resistance to antibiotics

Why do biofilm have a resistance for antibiotics?

1. Cells in deep layers may be metabolically inactive

2. EPS may slow diffusion of biocidal agents

3. Provide optimal environment for sharing of plasmids

What factors affect microbial growth?

1. Oxygen level

2. pH

3. Temperature

4. Osmotic pressure

5. Barometric pressure

What are some oxygen requirements for microbial growth?

O₂ is not always needed or tolerated, many enviroments do not have O₂

What are some names of oxygen requirements that can be used to group microbes?

• Obligate aerobes

• Obligate anaerobes

• Faculative anaerobes

• Aerotolerant anaerobes

• Microaerophiles

Wha does obligate stand for?

Must have oxygen

What does facultative stand for?

Can do both (can or not have oxygen)

What does aerotolerant stand for?

Tolerates oxygen

What does aerobe stand for?

Prefers oxygen

What does anaerobe stand for?

Prefers other than oxygen

What is fluid thioglycolate medium (FTM) stand for?

Low percentage agar tube that has a gradient of oxygen

How is aerotolerance determined?

Determined by location of growth

What is an example of obligate aerobes?

• Micrococcus lutes

• Pseudomonas aeruginosa

What are some examples of obligate anaerobes?

• Bacteroides spp.

• Clostridium botulinum

What are some examples of facultative anaerobes?

• Staphylococcus spp

• Escheria coli

What is an example of aerotolerant anaerobes?

Lactobacillus spp.

What are some examples of microaerophiles?

• Campylobacter spp.

• Neisseria gonorrhea

What are some pH requirements?

• pH can affect efficacy of macromolecules most vulnerable are proteins

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

• Fermenters are mostly adpated to acidity (think pickles)

• Organisms have min pH, optimal pH and max pH

What are the pH requirements for neutrophiles?

pH is 7

What are the pH requirements for acidophiles?

<5.5 pH

What are the pH requirements for alkaphiles?

pH 8-10.5

Do microbes have a optimal range for temperature?

Yes and also have a min and max temp

Temperature range for mesophiles?

20-40 Celcius

Temperature range for psychotrophs?

4-20 Celcius

Temperature range for Psychrophiles?

<0 Celcius

Temperature requirements for thermophiles?

50-80 Celcius

Temperature requirements for hyperthermophiles?

80-110 Celcius; some survive >112 Celcius

What is osmotic pressure?

Solute concentrations outside cell can have affect

• Halophiles and halotolerant

What are halophiles?

Salt/ solute lovers; found in oceans

What are halotolerant?

Tolerate high salt; salt marshes where high solutes arent present all the time (Recall: MSA and S. aureus)

What is basometric pressure?

• Ability to withstand great pressure

• Found on bottom of ocean

• Largelt unculturable; not much known

• Also can be thermo or hyperthermophiles

What are some nutrional requirements and media?

• Media can be selective to support growth of certain microbes

• Media can be further differential

• Based on salinity, fermentation, pH, etc

• Many require blood agar

What is alpha hemolysis (a)?

Hemoglobin is reduced causing browning

• Streptococcus pneumoniae

What is beta hemolysis (B)?

Complete lysis of RBC creating clearing zone

• Streptococcus pyogenes

What is gamma hemolysis (y)?

Growth but not hemolysis or reduction

• Enterococcus faecalis

What is selective media?

Contains nutrients that encourage growth of certain microbes

What is the mannitol salt media (MSA)?

Selects and differentiates pathogenic and non- pathogenic Staphylococci

What is MacConkey agar?

Selects and differentiates gram negative and lactose fermenters