BIOL 2460 Exam 3 UTA

Binary Fission

-most common form of bacterial reproduction

4 basic steps of bacterial reproduction

1. growth of cell size & increase in cell components

2. replication of DNA

3. division of the cytoplasm (cytokineses)

4. septum formation and division of daughter cells

Z ring assembly

1) Cytokinesis is directed by FtsZ protein

2) FtsZ assembles z-ring to form divisome

3) Divisome activates production of peptidoglycan and septum

generation time

-aka. doubling time

-time takes to double population

-varies greatly among species

calculating population size

-growth is exponential (if resources are no concern)

-population can be predicted from any starting size

Nn = N02^n

Nn = # of cells at generation n

n = # of generations

N0 = initial # of cells

-# of generation may need to be calculated

-Ex. generation time of 30 min = 48 generations in 24 hrs

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

culture density

# of cells per unit volume

Growth Curve

predictable pattern occurs

1. lag phase

2. log (exponential) phase

3. stationary phase

4. death phase

Growth Curve

lag phase

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

-initial cell #s do not change

-cells grow larger; metabolically active

-damaged or shocked cells undergo repair

-duration of phase determined by many factors

-genetic make-up

-media composition

-initial inoculum size

Growth Curve

log (exponential) phase

2. binary fission occurs; cell replication > cell death

-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 & antibiotics that affect protein, DNA, & cell-wall synthesis

Growth Curve

stationary phase

3. resources become depleted; cell replication = cell death

-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 & secondary metabolites

Growth Curve

death phase

4. endospores can form cell replication < cell death

-toxic waste accumulates; nutrients exhausted

-cells lyse & release nutrients for surviving cells & endospore-formers

-persisters- surviving cells with slow metabolism

~chronic infections (e.g. tuberculosis); antibiotic resistance

Growth Curve

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 to measure growth

-microscopic cell count

-fluorescent staining for alive & dead cells

-coulter count

-viable cell count

-optical density

Measuring Growth

direct microscopic cell count

-cells are counted under a microscope

-known volume is transferred to a calibrated slide (Petroff-Hausser chamber) & cells are manually counted

-cannot distinguish live vs. dead

Measuring Growth

fluorescence staining

-cells are counted under a microscope or flow cytometer

-red stain binds to damaged cells to indicate dead cells

Measuring Growth

coulter counter

-detects electrical resistance change due to cell density

-does NOT differentiate live/dead

Measuring Growth

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 & counted via pour plate or spread plate technique

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

~ <30 - TFTC

~ >300 - TNTC

-counting viable CFU requires distinguishable colonies

-achieved through serial dilution to achieve 30-300 CFU/ml range

-often dilutions are on log scale

-dilution "factor" is used to determine original CFU count

Measuring Growth

membrane filtration technique

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

-used on very dilute samples (e.g. drinking water) may not contain enough microbes for plate count

~sample concentrated instead of diluted

CD = CC/Vol

Measuring Growth

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

Measuring Growth

optical density (turbidity)

-measured with spectrophotometer

-light passed thru culture & 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 mainly in liquid environment (rivers, pipelines, oral cavity)

Biofilm Formation

biofilm structure

clusters of microbes in a matrix

Biofilm Formation - Biofilm Structure

extracellular polymeric substances (EPS)

-secreted by organisms in the biolfilm

~hydrated polysaccharide gel with other macromolecules & 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 through ____ ____, or cell to cell communication

-cell density or cellular stress

-autoinducer: small molecules are produced to induced various actions

quorum sensing

Biofilm and Human Health

-some are beneficial, some not

-biofilms often provide resistance to antibiotics

~cells in deep layers may be metabolically inactive

~EPS may slow diffusion of biocidal agents

~provide optimal environment for sharing of plasmids

Biofilm and Human Health

plaque formation on teeth is good or bad?

bad

Biofilm and Human Health

normal biota in lungs is good or bad?

good

Environmental Factors & Generation Time

for every extreme, there is likely a species

Environmental Factors & Generation Time

main factors that affect growth

-oxygen levels

-pH

-temperature

-osmotic pressure

-barometric pressure

Environmental Factors & Generation Time - Oxygen Requirements

is o2 always needed or tolerated?

no, many environments do not have 02 (anaerobic respiration)

Environmental Factors & Generation Time - Oxygen Requirements

optimal oxygen concentration

ideal concentration of O2

Environmental Factors & Generation Time - Oxygen Requirements

minimum permissive oxygen concentration

lowest O2 concentration allowing growth

Environmental Factors & Generation Time - Oxygen Requirements

maximum permissive oxygen concentration

highest O2 concentration allowing growth

Environmental Factors & Generation Time - Respiration Terminology

obligate

must have

Environmental Factors & Generation Time - Respiration Terminology

facultative

can do both

Environmental Factors & Generation Time - Respiration Terminology

aerotolerant

tolerant

Environmental Factors & Generation Time - Respiration Terminology

aerobe

prefers O2

Environmental Factors & Generation Time - Respiration Terminology

anaerobe

prefers others than O2

Environmental Factors & Generation Time - Respiration Terminology

-oxygen requirement can be used to group microbes

-obligate aerobes

-obligate anaerobes

-facultative anaerobes

-aerotolerant anaerobes

-microaerophiles

Environmental Factors & Generation Time - Oxygen Requirements

fluid thioglycolate medium (FTM)

low percentage agar tube has a gradient of oxygen

Environmental Factors & Generation Time - Oxygen Requirements

what determines aerotolerance?

location of growth

Environmental Factors & Generation Time - Oxygen Requirements

example of obligate aerobes

Micrococcus luteus

Environmental Factors & Generation Time - Oxygen Requirements

example of obligate anaerobe

Bacteroides spp.

Environmental Factors & Generation Time - Oxygen Requirements

example of facultative anaerobe

Staphylococcus spp.

Environmental Factors & Generation Time - Oxygen Requirements

example of aerotolerant anaerobe

Lactobacillus spp.

Environmental Factors & Generation Time - Oxygen Requirements

example of microaerophiles

Campylobacter spp.

anerobic jars or anaerobic chambers _____ o2

remove

Environmental Factors & Generation Time - pH Requirements

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

-microbes can prefer acidic of basic

-fermenters are mostly adapted to acidity (think pickles)

Environmental Factors & Generation Time - pH Requirements

optimal growth pH

most favorable pH for growth

Environmental Factors & Generation Time - pH Requirements

minimum growth pH

lowest pH for growth

Environmental Factors & Generation Time - pH Requirements

maximum growth pH

highest pH for growth

Environmental Factors & Generation Time - pH Requirements

groups by pH levels

-neutrophiles pH ~7

-acidophiles pH <5.5

-alkaliphiles pH 8-10.5

Environmental Factors & Generation Time - Temperature Requirements

groups by temp range

-mesophiles = 20-45 °C

-psychrotrophs = 4-20 °C

-psychrophiles = <0 °C

-thermophiles = 50-80 °C

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

Environmental Factors & Generation Time - Temperature Requirements

mesophiles temp

20-45 C

Environmental Factors & Generation Time - Temperature Requirements

psychrotrophs temp

4-20 C

Environmental Factors & Generation Time - Temperature Requirements

psychrophiles temp

<0 C

Environmental Factors & Generation Time - Temperature Requirements

Thermophiles temp

50-80 C

Environmental Factors & Generation Time - Temperature Requirements

hyperthermophiles temp

80-110 C; some >121 C

Environmental Factors & Generation Time - Osmotic Pressure & Growth

solute concentrations outside the cell can have effects

Environmental Factors & Generation Time - Osmotic Pressure & Growth

halophiles

salt/solute lovers; found in oceans

Environmental Factors & Generation Time - Osmotic Pressure & Growth

halotolerant

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

Environmental Factors & Generation Time - Barometric Pressure & Growth

ability to withstand great pressure

Environmental Factors & Generation Time - Barometric Pressure & Growth

barophiles

require high atmospheric pressure

-found on bottom of ocean

-largely unculturable; not much known

-can be thermo or hyperthermophiles

Environmental Factors & Generation Time - Light & Growth

photoautotrophs

cyanobacteria and green sulfurs

Environmental Factors & Generation Time - Light & Growth

photoheterotrophs

purple nonsulfurs

Environmental Factors & Generation Time - Light & Growth

photosynthetically active radiation (PAR)

usu. within visible light spectrum (400-700 nm)

Media Used for Bacterial Growth

all-purpose media (e.g. TSA)

Media Used for Bacterial Growth

enriched media

contains growth factors, vitamins, and other essentials to promote growth

~fastidous organisms - cannot make certain nutrients

Media Used for Bacterial Growth

chemically defined medium

complete chemical composition known

Media Used for Bacterial Growth

complex medium

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

Media Used for Bacterial Growth

selective media

inhibit unwanted, promote growth of organism of interest

Media Used for Bacterial Growth

enrichment cultures

promote growth of desired organism; only represents a fraction present

Media Used for Bacterial Growth

differential media

distinguish colonies of bacteria by color change

Controlling Microbial Growth

-clean ~ it's all relevant

-main goal: reduce microbial load & reduce infection or contamination

-not everything needs same level

~Sterilization

~Disinfection/Antisepsis

~Sanitation/Degerming

Controlling Microbial Growth

sterilization

removal/killing of ALL microbes

Controlling Microbial Growth

Disinfection/Antisepsis

inactivation of microbes

Controlling Microbial Growth

sanitation/degerming

decreasing microbial load

Biological Safety Level

-levels of cleanliness assigned to labs

-CDC, NIH, & WHO established 4 levels

Biological Safety Level Requirements

BSL-1

-very little risk

-sink for hand washing & door to close off lab

-agents that do not cause infection in healthy adults

Biological Safety Level Requirements

example of BSL-1

nonpathogenic E.coli and B. subtilis

(BIOL freshman labs)

Biological Safety Level Requirements

BSL-2

-pose moderate risk; restrictive access

-plus PPE, self-closing doors, eyewash station, autoclave or other sterilization method

Biological Safety Level Requirements

example of BSL-2

S. aureus & Salmonella spp.

~viruses like hepatitis, mumps & measles

(Micro labs)

Biological Safety Level Requirements

BSL-3

-potential to cause lethal infections by inhalation

-plus respirator, bio safety cabinets, hands free wash sink, two sets of doors, directional air flow

-indigenous or "exotic" pathogens

Biological Safety Level Requirements

example of BSL-3

M. tuberculosis & B. anthracis

west nile virus and HIV

Biological Safety Level Requirements

BSL-4

-most dangerous; often fatal

-plus full biohazard suit, change clothing on entry, shower on exit, decontaminate all material on exit, lab must have own air supply

-"exotic" pathogens

Biological Safety Level Requirements

example of bsl-4

ebola and marburg viruses

(only 13 in USA)

Level of Clean in the Clinic

critical

must be sterile; items used inside the body (i.e. sterile tissue or bloodstream)

Level of Clean in the Clinic

examples of critical

surgical instruments, catheters, IV fluids

Level of Clean in the Clinic

semicritical

do not require high level sterilization; items might contact non-sterile tissue (e.g. gut) but do not penetrate tissue

Level of Clean in the Clinic

example of semicritical

GI endoscope, respiratory therapy equipment

Level of Clean in the Clinic

noncritical

do not require sterilization; items contact but do not penetrate

Level of Clean in the Clinic

examples of noncritical

stethoscope, bed linens, blood pressure cuffs

Sterilization

complete killing or removal of all microbes from ______

fomite (inanimate objects)

Sterilization

several methods

-heat

-pressure

-filtration

-chemical (sterilants)

Sterilization

________ is used to prevent sterile environment from becoming contaminated

aseptic technique

Disinfection

disinfectant

inactivation/ kill of microbes on fomites

-some microbes may not be inactivated

disinfection ≠ sterile

Disinfection

disinfectant example

vinegar & bleach

Antisepsis

antiseptic

acts on microbes but not organisms/tissue