imn-02-pathogens in water and microbial growth

7/23/2019 IMN-02-Pathogens in Water and Microbial Growth

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Pathogens in Drinking water



Bacteria



Protists



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:



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



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

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



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.



Microbial Growth and

Metabolism



17

Growth Curve

Fig 5.4

pg 142

Living

Dead



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 adverseconditions

– antibiotics

– anti-microbial agents



Bacterial growth: exponential growth

Semilogarythmic plot

Straight line

indicateslogarithmic

growth



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



Bacterial growth: exponential growth

Generation time = 30 min

Cell volume = 5 mm3

.... 5 ml total cell volume

80 h7 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 massB. 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 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



dilutionsExample:

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




1. Concentration of a limiting nutrient

Results from a batch culture




2. Dilution rate



Factors affecting microbial growth

• Nutrients

• Temperature

• pH• Oxygen

• Water availability



Factors affecting microbial growth: Temperature

3 cardinal temperatures:

Usually ca. 30°C



„Temperature classes“ of organisms



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



Cyanobacteria growing

around Hot springs

Bacteria from ice cores

of Antartica



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)



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 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



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: 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



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



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

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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