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7/23/2019 IMN-02-Pathogens in Water and Microbial Growth
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Pathogens in Drinking water
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Bacteria
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Protists
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Indicator organisms
• Must be Present when pathogens are
present and be absent when pathogensare absent
• Must be easy to quantitatively analyse
• Must not be a serious pathogen itself• Must be present in the intestinal tract of
humans
Traits:
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Coliforms
• Present in colon
• Organisms that are under the genusEscherichia, Enterobacter , Klebsiella,Serratia, Citrobacter , and Proteus
• Collectively, this group of Gram-negativebacilli
• E. coli are ability to survive for briefperiods outside the body makes them anideal indicator organism for fecal
contamination
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Membrane filter technique
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Multiple Tube Fermentation
Technique
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Presumptive Test
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Presumptive test results
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Confirmation Test
• Brilliant green lactose bile broth
• Thermotolerant Fecal coliforms
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Completed Test
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Completed Test
E. coli vs E. aerogenes
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10mL tubes
Positive
1-mL tubes
positive
0.1 mL tubes
positive
MPN/100 mL
0 0 0 0
0 0 1 2
0 0 2 40 1 0 2
0 1 1 4
0 1 2 6
0 2 0 4
0 2 1 6
0 3 0 6
1 0 0 2
1 0 1 4
1 0 2 6
1 0 3 8
1 1 0 4
1 1 1 6
1 1 2 8
1 2 0 6
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Probability table
Combination MPN index/100mL
95% confidence limits
lower upper
0 0 0 <2
0 0 1 2 1 10
0 1 0 2 1 10
0 2 0 4 1 13
1 0 0 2 1 11
1 0 1 4 1 15
1 1 0 4 1 15
1 1 1 6 2 18
1 2 0 6 2 18
2 0 0 4 1 17
2 0 1 7 2 20
2 1 0 7 2 21
2 1 0 9 3 24
Interpretat ion : 95% o f the water samp les that
give this resul t contain 2 - 18 bacteria, w ith 6
being the mos t probable number.
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Microbial Growth and
Metabolism
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17
Growth Curve
Fig 5.4
pg 142
Living
Dead
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Phases of Growth
• 4 Phases
• 1. Lag Phase• 2. Log Phase
• 3. Stationary Phase
• 4. Death Phase
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1. Lag Phase
• Bacteria are first introduced into an
environment or media
• Bacteria are “checking out” their
surroundings
• cells are very active metabolically
• # of cells changes very little• 1 hour to several days
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2. Log Phase
• Rapid cell growth (exponential growth)
• population doubles every generation
• microbes are sensitive to adverseconditions
– antibiotics
– anti-microbial agents
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Bacterial growth: exponential growth
Semilogarythmic plot
Straight line
indicateslogarithmic
growth
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22
Rapid Generation Times
Fig 5.3
pg 140
1cell to 2 million cells
in 7 hours!
Only a build up of waste
or depletion of food will
stop growth
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Bacterial growth: exponential growth
Generation time = 30 min
Cell volume = 5 mm3
.... 5 ml total cell volume
80 h7 x 1036 m3 (> earth)
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3. Stationary Phase• Death rate = rate of reproduction
• cells begin to encounter environmental
stress
– lack of nutrients
– lack of water
– not enough space
– metabolic wastes
– oxygen
– pH
Endospores would form now
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4. Death Phase
• Death rate > rate of reproduction
• Due to limiting factors in the environment
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Measurement of microbial growth
A. Weight of cell massB. number of cells:
- Total cell count
- Viable count
- Dilutions
- turbidimetric
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total cell count
A. Sample dried on slide
B. Counting chamber:
Limitations:
- dead/live not distinguished
- small cells difficult to see
- precision low
- phase contrast microscope- not useful for < 106/ml
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viable cell countsynonymous: plate count, colony count
1 viable cell 1 colony
cfu = colony forming unitAdvantage: high sensitivity; selective media
Optimal: 30 – 300 colonies per plate ( plate appropriate dilutions)
spread plate method:
pour plate method:Bacteria must withstand 45°C briefly
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dilutionsExample:
3 h culture of E. coli in L-broth
How do I determine the actual number?
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Turbidimetric measurements
Relationship between OD and cfu/ml must be established experimentally
Exponential culture of E. coli in L-broth: 1 OD = ca. 2-3 x 109 cfu/ml
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Turbidimetric measurements
Limits of sensitivity at high bacterial density„rescattering“ more light reaches detector
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Continuous culture: the chemostat
steady state = cell number, nutrient status remain constant
Control:
1. Concentration of a limiting nutrient
2. Dilution rate
3. Temperature
Independent control of:- Cell density
- Growth rate
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Continuous culture: the chemostat
1. Concentration of a limiting nutrient
Results from a batch culture
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Continuous culture: the chemostat
2. Dilution rate
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Factors affecting microbial growth
• Nutrients
• Temperature
• pH• Oxygen
• Water availability
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Factors affecting microbial growth: Temperature
3 cardinal temperatures:
Usually ca. 30°C
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„Temperature classes“ of organisms
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Growth at high temperatures
<65°C
Thermophilic:
optimum > 45°C
Soil in sun often 50°C
Fermentation: 60-65°C
Hyperthermophilic:
optimum > 80°COnly in few areas:
Hot springs: 100°C
Steam vents 150-500°C
Deep sea hydrothermal vents
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Cyanobacteria growing
around Hot springs
Bacteria from ice cores
of Antartica
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Psychrophilic vs. Psychrotolerant
Psychrophiles
Maximum: <20°C
Optimum: <15°C
Minimum: <0°C
Habitats:
- deep sea- glaciers
Psychrotolerant
Maximum: >20°C
Optimum: 20-40°C
Minimum: <0-4°C
Habitats: much more abundant than
psychrophiles
- soil in temperate climate
- foods
- grow slowly even in fridge!
Sierra Nevada
Chlamydomonas nivalis
The snow algaered spores
Limit: Freezing
- Inhibits bacterial growth
- freezing: often liquid pockets
- many bacteria survive
- cryoprotectants (DMSO, glycerol)
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Bacterial growth: pH
(extremes: pH 4.6- 9.4)
Most
natural
habitats
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Bacterial growth: high pH
- Few alkaliphiles (pH10-11)- Bacteria: Bacillus spp.
- Archaea
- often also halophilic
- Sometimes: H+ gradient replaced by Na+ gradient (motility, energy)
- industrial applications (especially „exoenzymes“):
-Proteases/lipases for detergents (Bacillus licheniformis)-pH optima of these enzymes: 9-10
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Growth at low pH
Fungi: - often more acid tolerant
than bacteria (opt. pH5)
Obligate acidophilic bacteria:
Thiobacillus ferrooxidans
Obligate acidophilic Archaea:
Sulfolobus
Thermoplasma
Most critical: cytoplasmic membrane
Dissolves at more neutral pH
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Acid mine
drainage
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Bacterial growth: Osmosis
Water acitvity Osmotic pressure
aw =p
po
aw: rel. Water activity
p: vapor pressure of a solutionp0: vapor pressure of water
p =n x R x T
V
p: osmotic pressure
n: number of dissolved particles
R: universal gas constant
T: temperature
V: volume of the solution
low awhigh aw high p low p
Semipermeable membrane
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Bacterial growth: Osmosis
Soil: water activity = 0.9 – 1.0
In general: bacteria normally have higher osmotic pressure than environment= „positive water balance“
Osmophiles: - grow in presence of high sugar concentration
Xerophiles - grow in „dehydrated“ environments
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Bacterial growth: HalophilesHalophiles: - requirement for Na+
- grow optimally in media with low water activity- Mild: 1-6 % NaCl
- Moderate: 6-15 % NaCl
- extreme: 15 – 30% NaCl
most other organisms
would be dehydrated
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Bacterial growth at low aw: compatible solutes
Strategy: increase internal solute concentration
a. Pump inorganic ions
b. Synthesize organic solutesSolute must be „compatible“
with cellular processes
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Bacterial growth: Oxygen
O2 as electron sink for catabolism toxicity of Oxygen species
Aerobes: growth at 21% oxygen
Microaerophiles: growth at low oxygen concentration
Facultative aerobes: can grow in presence and absence of oxygen
Anaerobes: lack respiratory system
Aerotolerant anaerobes
Obligate anaerobes: cannot tolerate oxygen (lack of detoxification)
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Bacterial growth: Anaerobes
Methods to exclude/reduce oxygen:
- Closed vessels
- reducing agents (i.e. thioglycolate broth)
- anaerobic jar (H2-generation + Pd catalyst)
- glove box (oxygen free gas) a i r
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