Gim Lec Chapter 5

21.10.2012

LECTURE PRESENTA PRESENTATIONS TIONS

For BROCK BIOLOGY B IOLOGY OF MICROORGANISMS, THIRTEENTH THIRTEENTH EDITION Michael T. Madigan, Madigan, John M. Martinko, David A. Stahl, David P. Clark

Chapter 5

Microbial Growth Lectures by John Zamora Middle Tennessee State University © 2012 Pearson Education, Inc.

I. Bacterial Cell Division • 5.1 Cell Growth Growth and Binary Fission Fission

© 2012 Pearson Education, Inc.

Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI

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5.1 Cell Growth and Binary Fission • Growth: Growth: increase in the number of cells • Binary fission: fission: cell division following enlargement of a cell to twice its minimum size (Figure 5.1) • Generation time: time: time required for microbial cells to double in number • During cell division, each daughter cell receives a chromosome and sufficient copies copies of all other cell constituents to exist as an independent cell Animation: Overview Overview of Bacterial Growth

Animation: Binary Fission Fission © 2012 Pearson Education, Inc.


Figure 5.1 Binary fission in a rod-shaped prokaryote

Cell elongation n o i t a r e n e g e n O

Septum

Septum formation

Completion of septum; formation of walls; cell separation



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5.1 Cell Growth and Binary Fission • Growth: Growth: increase in the number of cells • Binary fission: fission: cell division following enlargement of a cell to twice its minimum size (Figure 5.1) • Generation time: time: time required for microbial cells to double in number • During cell division, each daughter cell receives a chromosome and sufficient copies copies of all other cell constituents to exist as an independent cell Animation: Overview Overview of Bacterial Growth

Animation: Binary Fission Fission © 2012 Pearson Education, Inc.


Figure 5.1 Binary fission in a rod-shaped prokaryote

Cell elongation n o i t a r e n e g e n O

Septum

Septum formation

Completion of septum; formation of walls; cell separation



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II. Population Growth • 5.5 The The Conc Concep eptt of of Exp Expon onen entia tiall Grow Growth th • 5.6 The The Mathe Mathema matic tics s of Expo Expone nenti ntial al Grow Growth th • 5.7 5.7 The The Mic Micro robi bial al Grow Growth th Cycl Cycle e • 5.8 Conti Continu nuous ous Cultu Culture: re: The The Chem Chemost ostat at



5.5 The Concept of Exponential Growth • Most bacteria have shorter generation times than eukaryotic microbes • Generation time is dependent on growth medium and incubation conditions



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5.5 The Concept of Exponential Growth • Exponential growth: growth of a microbial population in which cell numbers double within a specific time interval • During exponential growth, the increase in cell number is initially slow but increases at a faster rate (Figure 5.8)



Figure 5.8 The rate of growth of a microbial culture

) 1000 e l s Logarithmic l l a e c plot c s f i c o t Arithmetic r e e m 500 plot b h t m i u r a N (

103 102

10

) e l a s l l c e s c c f i o m r h e t i b r a m g u l o N (

100 0


1

3 4 2 Time (h)

5

1


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5.6 The Mathematics of Exponential Growth • A relationship exists between the initial number of cells present in a culture and the number present after a period of exponential growth: N = N 0 2n N is the final cell number N 0 is the initial cell number n is the number of generations during the period of exponential growth



5.6 The Mathematics of Exponential Growth • Generation time (g ) of the exponentially growing population is g = t/n t is the duration of exponential growth n is the number of generations during the period of exponential growth



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5.6 The Mathematics of Exponential Growth • Specific growth rate (k ) is calculated as

k



Slope

log(2) 

2



0.15

k = 0.301/g • Division rate (v ) is calculated as v = 1/g Figure 5.9 Calculating microbial growth parameters. Method of estimating the generation times (g) of exponentially growing populations with generation times of 2 h from data plotted on semilogarithmic graphs. The slope of line is equal to 0.30/g, and n is the number of generations in the time t. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI


5.7 The Microbial Growth Cycle • Batch culture: a closed-system microbial culture of fixed volume • Typical growth curve for population of cells grown in a closed system is characterized by four phases (Figure 5.10): – Lag phase – Exponential phase – Stationary phase – Death phase Animation: Bacterial Growth Curve © 2012 Pearson Education, Inc.


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Figure 5.10 Typical growth curve for a bacterial population

Growth phases Lag

Exponential

Stationary

Death 1.0

10

0.75

l m e / l b s 9 a m i s v i 0 n 1 a g g o r L o 8

Turbidity (optical density)

0.50

Viable count

) D O ( y t i s n e d l a c i t p O

0.25 7

6 Time

0.1

A viable count measures the cells in the culture that are capable of reproducing. Optical density (turbidity), a quantitative measure of light scattering by a liquid culture, increases with the increase in cell number. © 2012 Pearson Education, Inc.


5.7 The Microbial Growth Cycle • Lag phase – Interval between when a culture is inoculated and when growth begins

• Exponential phase – Cells in this phase are typically in the healthiest state

• Stationary phase – Growth rate of population is zero – Either an essential nutrient is used up or waste product of the organism accumulates in the medium © 2012 Pearson Education, Inc.


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5.7 The Microbial Growth Cycle • Death Phase – If incubation continues after cells reach stationary phase, the cells will eventually die



5.8 Continuous Culture: The Chemostat • Continuous culture: an open-system microbial culture of fixed volume • Chemostat : most common type of continuous culture device (Figure 5.11) – Both growth rate and population density of culture can be controlled independently and simultaneously • Dilution rate: rate at which fresh medium is pumped in and spent medium is pumped out (mean cell residence time or hydraulic retention time ‘HRT’ ) • Concentration of a limiting nutrient © 2012 Pearson Education, Inc.


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Figure 5.11 Schematic for a continuous culture device (chemostat)

Fresh medium from reservoir

Flow-rate regulator

Sterile air or other gas

Gaseous headspace Culture vessel Culture Overflow

Effluent containing microbial cells

The population density is controlled by the concentration of limiting nutrient in the reservoir, and the growth rate is controlled by the flow rate. Both parameters can be set by the experimenter. © 2012 Pearson Education, Inc.


5.8 Continuous Culture: The Chemostat • In a chemostat – The growth rate is controlled by dilution rate – The growth yield (cell number/ml) is controlled by the concentration of the limiting nutrient

• In a batch culture, growth conditions are constantly changing; it is impossible to independently control both growth parameters (Figure 5.12)



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Figure 5.12 The effect of nutrients on growth

Rate and yield affected

Only yield affected

)

)

( e t a r h t w o r G

( d l e i y h t w o r G

0

0.1

0.2

0.3

0.4

0.5

Nutrient concentration (mg/ml)

Relationship between nutrient concentration, growth rate (green curve), and growth yield (red curve) in a batch culture (closed system). Only at low nutrient concentrations are both growth rate and growth yield affected. © 2012 Pearson Education, Inc.


5.8 Continuous Culture: The Chemostat • Chemostat cultures are sensitive to the dilution rate and limiting nutrient concentration (Figure 5.13) – At too high a dilution rate, the organism is washed out – At too low a dilution rate, the cells may die from starvation – Increasing concentration of a limiting nutrient results in greater biomass but same growth rate



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Figure 5.13 Steady-state relationships in the chemostat

) l / g ( n o i t a r t n e c n o c l a i r e t c a b e t a t s y d a e t S

Steady state 5

Bacterial concentration 6

4

3

4

2 2

) h ( e m i t g n i l b u o D

1

0

0 0

0.25

0.5 Dilution rate (h

0.75 1)

1.0

Washout

At high dilution rates, growth cannot balance dilution, and the population washes out. Although the population density remains constant during steady state, the growth rate (doubling time) can vary over a wide range. © 2012 Pearson Education, Inc.


III. Measuring Microbial Growth • 5.9 Microscopic Counts • 5.10 Viable Counts • 5.11 Turbidimetric Methods



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5.9 Microscopic Counts • Microbial cells are enumerated by microscopic observations (Figure 5.14) – Results can be unreliable



Figure 5.14 Direct microscopic counting procedure using the Petroff –Hausser counting chamber



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5.9 Microscopic Counts • Limitations of microscopic counts – Cannot distinguish between live and dead cells without special stains – Small cells can be overlooked – Precision is difficult to achieve – Phase-contrast microscope required if a stain is not used – Cell suspensions of low density (<106 cells/ml) hard to count – Motile cells need to immobilized – Debris in sample can be mistaken for cells © 2012 Pearson Education, Inc.


5.9 Microscopic Counts • A second method for enumerating cells in liquid samples is with a flow cytometer – Uses laser beams, fluorescent dyes, and electronics



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5.10 Viable Counts • Viable cell counts (plate counts): measurement of living, reproducing population – Two main ways to perform plate counts: • Spread-plate method (Figure 5.15) • Pour-plate method

• To obtain the appropriate colony number, the sample to be counted should always be diluted (Figure 5.16)



Figure 5.15 Two methods for the viable count

Spread-plate method Surface colonies

Incubation

Sample is pipetted onto surface of agar plate (0.1 ml or less)

Sample is spread evenly over surface of agar using sterile glass spreader

Typical spread-plate results

Pour-plate method

Colonies of Es cherichiacoli

formed from cells plated by the spreadplate method (top) or the pour-plate method (bottom)

Surface colonies

Solidification and incubation

Sample is pipetted into sterile plate


Sterile medium is added and mixed well with inoculum

Subsurface colonies Typical pour-plate results

Colonies form within the agar as well as on the agar surface


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Figure 5.16 Procedure for viable counting using serial dilutions of the sample and the pour-plate method

The sterile liquid used for making dilutions can simply be water, but a solution of mineral salts or actual growth medium may yield a higher recovery.

Sample to be counted

1 ml

1 ml

1 ml

1 ml

1 ml

1 ml

9-ml broth

Total 1/10 dilution (10 1)

1/100 (10 2)

1/103 (10 3)

1/10 4 (10 4)

1/10 5 (10 5)

1/10 6 (10 6)

Plate 1-ml samples

Too many colonies to count

159 17 2 0 colonies colonies colonies colonies

159 103 Plate Dilution count factor © 2012 Pearson Education, Inc.

1.59 105 Cells (colony-forming units) per milliliter o f original sample Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI

5.10 Viable Counts • Plate counts can be highly unreliable when used to assess total cell numbers of natural samples (e.g., soil and water) – Selective culture media and growth conditions target only particular species



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5.10 Viable Cell Counting • The Great Plate Anomaly : direct microscopic counts of natural samples reveal far more organisms than those recoverable on plates • Why is this? – Microscopic methods count dead cells whereas viable methods do not – Different organisms may have vastly different requirements for growth



5.11 Turbidimetric Methods • Turbidity measurements are an indirect, rapid, and useful method of measuring microbial growth (Figure 5.17a) – Most often measured with a spectrophotometer and measurement referred to as optical density (O.D.)



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Figure 5.17a Turbidity measurements of microbial growth

Light

Prism Incident light, I 0 Filter

Measurements of turbidity are made in a spectrophotometer. The photocell measures incident light unscattered by cells in suspension and gives readings in optical density units.

Sample containing cells ( ) Unscattered light, I Photocell (measures unscattered light, I ) Spectrophotometer Optical density (OD) Log


I 0 I


5.11 Turbidimetric Methods • To relate a direct cell count to a turbidity value, a standard curve must first be established (Figure 5.17c)



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Figure 5.17c Turbidity measurements of microbial growth

0.8 y 0.7 t i s n 0.6 e d l 0.5 a c i t 0.4 p O

Theoretical Actual

The one-to one correspondence between these relationships breaks down at high turbidities

0.3

0.2 0.1 Cell numbers or mass (dry weight)

Relationship between cell number or dry weight and turbidity readings. © 2012 Pearson Education, Inc.


5.11 Turbidimetric Methods • Turbidity measurements – Quick and easy to perform – Typically do not require destruction or significant disturbance of sample – Sometimes problematic (e.g., microbes that form clumps or biofilms in liquid medium)



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IV. Temperature and Microbial Growth • 5.12 Effect of Temperature on Growth • 5.13 Microbial Life in the Cold • 5.14 Microbial Life at High Temperatures



5.12 Effect of Temperature on Growth • Temperature is a major environmental factor controlling microbial growth • Cardinal temperatures: the minimum, optimum, and maximum temperatures at which an organism grows (Figure 5.18)



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Figure 5.18 The cardinal temperatures: minimum, optimum, and maximum

Enzymatic reactions occurring at maximal possible rate

Optimum e Enzymatic reactions occurring t a r at increasingly rapid rates h t w o r G

Minimum

Maximum

Actual values may vary greatly for different organisms

Temperature Membrane gelling; transport processes so slow that growth cannot occur © 2012 Pearson Education, Inc.

Protein denaturation; collapse of the cytoplasmic membrane; thermal lysis


5.12 Effect of Temperature on Growth • Microorganisms can be classified into groups by their growth temperature optima (Figure 5.19) – Psychrophile: low temperature – Mesophile: midrange temperature – Thermophile: high temperature – Hyperthermophile: very high temperature



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Figure 5.19 Temperature and growth response in different temperature classes of microorganisms

Thermophile Example:

Hyperthermophile G eobacillus stearothermophilus Example: Thermococcus celer

Mesophile Example:

60

e E scheric hia coli t a r h 39 t Psychrophile w Example: o r Polaromonas vacuolata G

Hyperthermophile Example:

Pyr olobus fumarii

106

88

4

0

10

20

30

40

50

60

70

80

90

100

110

120

Temperature (oC)



5.12 Effect of Temperature on Growth • Mesophiles: organisms that have midrange temperature optima; found in – Warm-blooded animals – Terrestrial and aquatic environments – Temperate and tropical latitudes



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5.13 Microbial Life in the Cold • Extremophiles – Organisms that grow under very hot or very cold conditions

• Psychrophiles – Organisms with cold temperature optima – Inhabit permanently cold environments (Figure 5.20)

• Psychrotolerant – Organisms that can grow at 0ºC but have optima of 20ºC to 40ºC – More widely distributed in nature than psychrophiles © 2012 Pearson Education, Inc.


5.13 Microbial Life in the Cold • Molecular Adaptations to Psychrophily – Production of enzymes that function optimally in the cold; features that may provide more flexibility • More -helices than -sheets • More polar and less hydrophobic amino acids • Fewer weak bonds • Decreased interactions between protein domains



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5.13 Microbial Life in the Cold • Molecular Adaptations to Psychrophily (cont’d) – Transport processes function optimally at low temperatures • Modified cytoplasmic membranes – High unsaturated fatty acid content



5.14 Microbial Life at High Temperatures • Above ~65 oC, only prokaryotic life forms exist • Thermophiles: organisms with growth temperature optima between 45 oC and 80 oC • Hyperthermophiles: organisms with optima greater than 80 oC – Inhabit hot environments including boiling hot springs and seafloor hydrothermal vents that can have temperatures in excess of 100 oC



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5.14 Microbial Life at High Temperatures • Hyperthermophiles in Hot Springs – Chemoorganotrophic and chemolithotrophic species present (Figure 5.22) – High prokaryotic diversity (both Archaea and Bacteria represented)



Figure 5.22 Growth of hyperthermophiles in boiling water

(a) Boulder Spring, a small boiling spring in Yellowstone National Park. This spring is superheated, having a temperature 1-2 oC above the boiling point. The mineral deposits around the spring consist mainly of silica and sulfur. (b) Photomicrograph of a microcolony of prokaryotes that developed on a microscope slide immersed in such a boiling spring. © 2012 Pearson Education, Inc.


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5.14 Microbial Life at High Temperatures • Studies of thermal habitats have revealed – Prokaryotes are able to grow at higher temperatures than eukaryotes – Organisms with the highest temperature optima are Archaea – Nonphototrophic organisms can grow at higher temperatures than phototrophic organisms



5.14 Microbial Life at High Temperatures • Molecular Adaptations to Thermophily – Enzyme and proteins function optimally at high temperatures; features that provide thermal stability • Critical amino acid substitutions in a few locations provide more heat-tolerant folds • An increased number of ionic bonds between basic and acidic amino acids resist unfolding in the aqueous cytoplasm • Production of solutes (e.g., di-inositol phophate, diglycerol phosphate) help stabilize proteins



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5.14 Microbial Life at High Temperatures • Molecular Adaptations to Thermophily (cont’d) – Modifications in cytoplasmic membranes to ensure heat stability • Bacteria have lipids rich in saturated fatty acids • Archaea have lipid monolayer rather than bilayer



V. Other Environmental Factors Affecting Growth • 5.15 Acidity and Alkalinity • 5.16 Osmotic Effects on Microbial Growth • 5.17 Oxygen and Microorganisms • 5.18 Toxic Forms of Oxygen



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5.15 Acidity and Alkalinity • The pH of an environment greatly affects microbial growth (Figure 5.24) • Some organisms have evolved to grow best at low or high pH, but most organisms grow best between pH 6 and 8 (neutrophiles)



Figure 5.24 The pH scale

pH Example

Moles per liter of: OH

H 1

s e l i h p o Increasing d i acidity c A

Neutrality

s e l i h p i l a k l A

Increasing alkalinity


10

14

Volcanic soils, waters Gastric fluids Lemon juice Acid mine drainage Vinegar Rhubarb Peaches

10

1

10

13

10

2

10

12

10

3

10

11

Acid soil Tomatoes American cheese Cabbage Peas Corn, salmon, shrimp

10

4

10

10

10

5

10

9

10

6

10

8

7

7

10

Pure water

10

Seawater

10

8

10

6

Very alkaline natural soil Alkaline lakes Soap solutions Household ammonia Extremely alkaline soda lakes Lime (saturated solution)

10

9

10

5

10

10

10

4

10

11

10

3

10

12

10

2

10

13

10

1

10

14

1

Although some microorganisms can live at very low or very high pH, the cell’s internal pH remains near neutrality.


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5.15 Acidity and Alkalinity • Acidophiles: organisms that grow best at low pH (<6) – Some are obligate acidophiles; membranes destroyed at neutral pH – Stability of cytoplasmic membrane critical

• Alkaliphiles: organisms that grow best at high pH (>9) – Some have sodium motive force rather than proton motive force



5.15 Acidity and Alkalinity • The internal pH of a cell must stay relatively close to neutral even though the external pH is highly acidic or basic – Internal pH has been found to be as low as 4.6 and as high as 9.5 in extreme acido- and alkaliphiles, respectively

• Microbial culture media typically contain buffers to maintain constant pH



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5.16 Osmotic Effects on Microbial Growth • Water activity (aw ): water availability; expressed in physical terms – Defined as ratio of vapor pressure of air in equilibrium with a substance or solution to the vapor pressure of pure water



5.16 Osmotic Effects on Microbial Growth • Typically, the cytoplasm has a higher solute concentration than the surrounding environment, thus the tendency is for water to move into the cell ( positive water balance) • When a cell is in an environment with a higher external solute concentration, water will flow out unless the cell has a mechanism to prevent this



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5.16 Osmotic Effects on Microbial Growth • Halophiles: organisms that grow best at reduced water potential; have a specific requirement for NaCl (Figure 5.25) • Extreme halophiles: organisms that require high levels (15 –30%) of NaCl for growth • Halotolerant : organisms that can tolerate some reduction in water activity of environment but generally grow best in the absence of the added solute Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI


Figure 5.25 Effect of sodium chloride (NaCl) concentration on growth of microorganisms of different salt tolerances or requirements.

Halotolerant Halophile

Example:

Example:

S taphyloc occus aureus

Extreme halophile

A lii vibri o fis cheri Example: Halobacterium s alinar um

e t a r h t w o r G

Nonhalophile Example:

E scherichia coli 0

5

10

15

20

NaCl %

Optimum NaCl concentration for extreme halophiles, it is between 15-30% © 2012 Pearson Education, Inc.


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5.16 Osmotic Effects on Microbial Growth • Osmophiles: organisms that live in environments high in sugar as solute • Xerophiles: organisms able to grow in very dry environments



5.16 Osmotic Effects on Microbial Growth • Mechanisms for combating low water activity in surrounding environment involve increasing the internal solute concentration by – Pumping inorganic ions from environment into cell – Synthesis or concentration of organic solutes • compatible solutes: compounds used by cell to counteract low water activity in surrounding environment



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5.17 Oxygen and Microorganisms • Aerobes: require oxygen to live • Anaerobes: do not require oxygen and may even be killed by exposure • Facultative organisms: can live with or without oxygen • Aerotolerant anaerobes: can tolerate oxygen and grow in its presence even though they cannot use it • Microaerophiles: can use oxygen only when it is present at levels reduced from that in air © 2012 Pearson Education, Inc.


5.17 Oxygen and Microorganisms • Thioglycolate broth (Figure 5.26) – Complex medium that separates microbes based on oxygen requirements – Reacts with oxygen so oxygen can only penetrate the top of the tube



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Figure 5.26 Growth versus oxygen (O 2) concentration

The redox dye, resazurin, which is pink when oxidized and colorless when reduced, has been added as a redox indicator.

s e b o r e A

s e b o r e a n A

s e b o r e a e v i t a t l u c a F


s e l i h p o r e a o r c i M

s e b o r e a n a t n a r e l o t o r e A

(a) O2 penetrates only a short distance into the tube, so obligate aerobes grow only close to the surface. (b) Anaerobes, being sensitive to O2, grow only away from the surface. (c) Facultative aerobes are able to grow in either the presence or Oxic zone the absence of O 2 and thus grow throughout the tube. However, growth is better near the surface because these Anoxic zone organisms can respire. (d) Microaerophiles grow away from the most oxic zone. (e) Aerotolerant anaerobes grow throughout the tube. Growth is not better near the surface because these organisms can only ferment. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI

5.17 Oxygen and Microorganisms • Special techniques are needed to grow aerobic and anaerobic microorganisms (Figure 5.27) • Reducing agents: chemicals that may be added to culture media to reduce oxygen (e.g., thioglycolate)



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Figure 5.27 Incubation under anoxic conditions

Anoxic jar

Anoxic glove bag

A chemical reaction in the envelope in the jar generates H2 + CO2. The H2 reacts with O2 in the jar on the surface of a palladium catalyst to yield H2O; the final atmosphere contains N2, H2, and CO2.

Anoxic glove bag for manipulating and incubating cultures under anoxic conditions. The airlock on the right, which can be evacuated and filled with O2-free gas, serves as a port for adding and removing materials to and from the glove bag.



5.18 Toxic Forms of Oxygen • Several toxic forms of oxygen can be formed in the cell (Figure 5.28): – Single oxygen – Superoxide anion – Hydrogen peroxide – Hydroxyl radical



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Gim Lec Chapter 5

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