Pathogens in Drinking water

Bacteria

Protists

Indicator organisms
Traits:

Must be Present when pathogens are

present and be absent when pathogens
are absent
Must be easy to quantitatively analyse
Must not be a serious pathogen itself
Must be present in the intestinal tract of
humans

Coliforms
Present in colon
Organisms that are under the genus
Escherichia, Enterobacter, Klebsiella,
Serratia, Citrobacter, and Proteus
Collectively, this group of Gram-negative
bacilli
E. coli are ability to survive for brief
periods outside the body makes them an
ideal indicator organism for fecal
contamination

Membrane filter technique

Multiple Tube Fermentation

Technique

Presumptive Test

Presumptive test results

Confirmation Test
Brilliant green lactose bile broth
Thermotolerant Fecal coliforms

Completed Test

Completed Test

E. coli vs E. aerogenes

10mL tubes
Positive

1-mL tubes
positive

0.1 mL tubes
positive

MPN/100 mL

Probability table
95% confidence limits
Combination

MPN index/100mL

lower

upper

<2

10

10

13

11

15

15

18

18

17

20

21

24

Interpretation: 95% of the water samples that

give this result contain 2 - 18 bacteria, with 6
being the most probable number.

Microbial Growth and

Metabolism

Growth Curve

Living
Dead

Fig 5.4
pg 142

17

Phases of Growth
4 Phases

1. Lag Phase
2. Log Phase
3. Stationary Phase
4. Death Phase

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

2. Log Phase
Rapid cell growth (exponential growth)
population doubles every generation
microbes are sensitive to adverse
conditions
antibiotics
anti-microbial agents

Bacterial growth: exponential growth

Semilogarythmic plot

Straight line
indicates
logarithmic
growth

Rapid Generation Times

1cell to 2 million cells

in 7 hours!
Only a build up of waste
or depletion of food will
stop growth
Fig 5.3
pg 140

22

Bacterial growth: exponential growth

Generation time = 30 min

Cell volume = 5 mm3
.... 5 ml total cell volume
80 h

7 x 1036 m3 (> earth)

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

4. Death Phase
Death rate > rate of reproduction
Due to limiting factors in the environment

Measurement of microbial growth

A. Weight of cell mass
B. number of cells:
- Total cell count
- Viable count
- Dilutions
- turbidimetric

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

viable cell count

synonymous: plate count, colony count
1 viable cell 1 colony
cfu = colony forming unit
Advantage: high sensitivity; selective media
Optimal: 30 300 colonies per plate ( plate appropriate dilutions)
spread plate method:

pour plate method:

Bacteria must withstand 45C briefly

dilutions
Example:
3 h culture of E. coli in L-broth
How do I determine the actual number?

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

Turbidimetric measurements

Limits of sensitivity at high bacterial density

rescattering more light reaches detector

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

Continuous culture: the chemostat

1. Concentration of a limiting nutrient

Results from a batch culture

Continuous culture: the chemostat

2. Dilution rate

Factors affecting microbial growth

Nutrients
Temperature
pH
Oxygen
Water availability

Factors affecting microbial growth: Temperature

3 cardinal temperatures:

Usually ca. 30C

Temperature classes of organisms

Growth at high temperatures

Thermophilic:
optimum > 45C
Soil in sun often 50C
Fermentation: 60-65C

<65C

Hyperthermophilic:
optimum > 80C
Only in few areas:
Hot springs: 100C
Steam vents 150-500C
Deep sea hydrothermal vents

Cyanobacteria growing
around Hot springs

Bacteria from ice cores

of Antartica

Psychrophilic vs. Psychrotolerant

Sierra Nevada
Psychrophiles
Maximum: <20C
Optimum: <15C
Minimum: <0C
Habitats:
- deep sea
- glaciers

red spores

Chlamydomonas nivalis
The snow algae

Psychrotolerant
Maximum: >20C
Optimum: 20-40C
Minimum: <0-4C
Habitats: much more abundant than
psychrophiles
- soil in temperate climate
- foods
- grow slowly even in fridge!

Limit: Freezing
- Inhibits bacterial growth
- freezing: often liquid pockets
- many bacteria survive
- cryoprotectants (DMSO, glycerol)

Bacterial growth: pH

(extremes: pH 4.6- 9.4)

Most
natural
habitats

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

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

Acid mine
drainage

Bacterial growth: Osmosis

Water acitvity
aw =

p
po

aw: rel. Water activity

p: vapor pressure of a solution
p0: vapor pressure of water

Osmotic pressure
p=

nxRxT
V

p: osmotic pressure
n: number of dissolved particles
R: universal gas constant
T: temperature
V: volume of the solution

Semipermeable membrane

high aw

low aw

low p

high p

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

Bacterial growth: Halophiles

Halophiles:

- requirement for Na+

- grow optimally in media with low water activity
- Mild: 1-6 % NaCl
most other organisms
- Moderate: 6-15 % NaCl
would be dehydrated
- extreme: 15 30% NaCl

Bacterial growth at low aw: compatible solutes

Strategy: increase internal solute concentration
a. Pump inorganic ions
b. Synthesize organic solutes

Solute must be compatible

with cellular processes

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)

Bacterial growth: Anaerobes

air

air

air

air

air

obl. anaerobe

fac. aerobe

microaerophile

aerotolerant
anaerobe

- Closed vessels
- reducing agents (i.e. thioglycolate broth)
- anaerobic jar (H2-generation + Pd catalyst)
- glove box (oxygen free gas)

obl. aerobe

Methods to exclude/reduce oxygen:

O2