chapter 3

Shixue Yin (Prof Dr)CESE, Yangzhou University

Chapter 3

Microbial Growth


Microbial Growth means

increase in number of cells, not cell size. It is very confusing with reproduction ( 繁殖 ) in other forms of life (e. g. animals).


Growth requirements

Physical

Chemical

1 Temperature

2 pH

3 Osmotic pressure (water)4 Light

1 Carbon

2 Nitrogen

3 Sulfur4 Phosphorus

5 Inorganic element

6 Oxygen

7 growth factors


•Temperature–Minimum growth temperature

–Optimum growth temperature

–Maximum growth temperature

Physical Requirements for Growth: 1 temperature


专性嗜冷兼性嗜冷中温型嗜热极端嗜热

Arctic/antarctic Sea/refrige

mammal/soils compost

Spring/sea volcano


Question:

Is it always safe if foods are stored in refrigerator for long time?


Figure 6.2


• pH– Most bacteria grow between pH

6.5 and 7.5– Molds and yeasts grow between

pH 5 and 6– Acidophiles grow in acidic

environments

Physical Requirements for Growth: 2 pH

Shixue Yin (Prof Dr)CESE, Yangzhou UniversityPhysical Requirements for

Growth: 3 osmotic pressure

• Osmotic Pressure (=water availability)

– Hypertonic ( 高渗 ) environments, increase salt or sugar, cause plasmolysis( 质壁分离 )

– Extreme or obligate ( 专性 ) halophiles require high osmotic pressure

– Facultative ( 兼性 ) halophiles tolerate high osmotic pressure


Plasmolysis

Figure 6.4

Cell in normal osmotic pressure environment

Cell in hypertonic

environment


Physical Requirements for Growth: 4 Light

• Light (=radiation)

– Necessary for phototrophic bacteria (using light as energy source)

– Radiation in different wavelength has different effects (ultraviolet, x-ray, and gamma-ray kills bacteria while green light induces development of life cycle, e. g. mushrooms)


• Carbon

– Structural organic molecules, energy source (recall the chemical constituents of cells)

– Chemoheterotrophs ( 化能异养型 ) use organic carbon sources

– Autotrophs ( 自养型 ) use CO2

Chemical Requirements for Growth: 1 carbon


1. NitrogenA. In amino acids, proteinsB. Most bacteria decompose proteins

C. Some bacteria use NH4+ or NO3

D. A few bacteria use N2 in nitrogen fixation

2. SulfurA. In amino acids, thiamine ( 硫胺素 =Vb 1),

biotin ( 生物素 VH)B. Most bacteria decompose proteins

C. Some bacteria use SO42 or H2S

Chemical Requirements for Growth:


3. Phosphorus

A. In DNA, RNA, ATP, and membranes

B. PO43 is a source of phosphorus

4. Trace elements

A. Inorganic elements required in small amounts

B. Usually as enzyme cofactors

Chemical Requirements for Growth


5. Oxygen (O2)


Obligate aerobes

专性需氧菌

O2 is necessar

y

Facultative anaerobes兼性厌氧菌

Grow better if

O2 is present

Obligate anaerobes专性厌氧菌

O2 is toxic

Aerotolerant anaerobes

微耐氧菌

O2 is not necessary

but tolerable

Micro-aerophiles

微需氧菌

O2 is needed at low partial

pressure


• Singlet oxygen: O2 boosted to a higher-energy state

• Superoxide free radicals ( 超氧化物自由基 ): O2

• Peroxide anion ( 过氧化物阴离子 ): O22

• Hydroxyl radical 羟基 (OH)

Why O2 is toxic to some bacteria

Some bacteria do not have these enzymes


6. Organic Growth Factors

A. Organic compounds obtained from the environment

B. Vitamins, amino acids, purines ( 嘌呤 ), pyrimidines ( 嘧啶 )



• Culture Medium: Nutrients prepared for microbial growth

• Sterile: No living microbes

• Inoculum: Introduction of microbes into medium

• Culture: Microbes growing in/on culture medium

Culture Media


• Complex polysaccharide

• Used as solidifying agent for culture media in Petri plates ( 平板 ), slants (斜面 ), and deeps ( 深层培养 )

• Generally not metabolized by microbes

• Liquefies at 100°C

• Solidifies ~40°C

Agar


• Chemically defined media: exact chemical composition is known

• Complex media: Extracts and digests of yeasts, meat, or plants, e. g.

– Nutrient broth ( 营养肉汤 )

– Nutrient agar ( 营养琼脂 )

Culture Media


Examples of Culture Media


• Reduced media

– Contain chemicals (thioglycollate 巯基乙酸盐 or cystine ( 胱氨酸 ) or ascorbate ( 抗坏血酸 ) to remove O2

– Heated to drive off O2

Anaerobic Culture Methods


• Anaerobic jar

Anaerobic Culture Methods

钯


• Anaerobic chamber

Anaerobic

Culture Methods


• Candle jar O2-packet

Some bacteria require high CO2


• Enhance the growth of certain wanted organisms but suppress unwanted microbes.

Selective Media

Figure 6.9b, c

• Make it easy to distinguish colonies of different microbes.

Differential Media


• Encourages growth of desired microbe

• Assume a soil sample contains a few phenol-degrading bacteria and thousands of other bacteria

– Inoculate phenol-containing culture medium with the soil and incubate

– Transfer 1 ml to another flask of the phenol medium and incubate

– Transfer 1 ml to another flask of the phenol medium and incubate

– Only phenol-metabolizing bacteria will be growing

Enrichment Media


•An all-purposed (rich) medium is rich in a wide variety of nutrients (including many growth factors) and will, therefore, support the growth of a wide range of bacteria.

All-purposed (rich) medium


•A Minimal medium supplies only the minimal nutritional requirements of a particular organism.

Minimal Medium


Summary of Commonly-Used Constituents of Media

•Substance Function Composition Source

•AGAR Solidifying agent Impure polysaccharide marine algae

•PEPTONES nutrient Animal/Plant proteins cow,soy

•EXTRACTS nutrient Animal/Bacteria paste cow,yeast

•BODY FLUIDS hormones Blood animals

•BUFFERS pH K2PO4;NaHPO3;CaCO3 -

•REDUCTANTS e- source thioglycolate -

•SELECTIVES bacteriostat Antibiotics, sodium azide varies

•INDICATORS pH bromothymol blue,phenol red -

•WATER hydration H2O (DI & tap) -


• A pure culture contains only one species or strain

• A colony is a population of cells arising from a single cell or spore or from a group of attached cells

• A colony is often called a colony-forming unit (CFU)

A few words


Streak Plate ( 平板划线 )


Medium sterilization


Sterilization: all living cells, viable spores, viruses are killed or removed from object or habitat though:

Irradiation: Irradiation: destroys/distorts nucleic acids X-rays & microwaves. UV commonly used on object surfaces

Filtration:Filtration: physical removal from liquid or gas. Sterilize soln’s that are denatured by heat, i.e., antibiotics, injectable drugs, amino acids, vitamins, etc

Gas:Gas: formaldehyde, glutaraldehyde, ethylene oxide. Toxic chemicals (require gas chamber) used for large items

Heat:Heat: important, widely used. Endospores (theromoduric) destruction guarantees sterility


Treatment

Temp EffectivenessIncineratio

n>500o Vaporizes organic material

on non-flammable surfaces but may destroy many substances in the process

Boiling 100o 30 minutes boiling kills microbial pathogens & vegetative forms of bacteria BUT may not kill endospores

Intermittent Boiling

100o 3x30 min intervals, followed by periods of cooling kills endospores


Autoclave/pressure cooker (steam under pressure)

121o/15mins @ 15lbs pressure

Kills all forms of life including endospores. Sterilization requires maintenance at effective temp for full time period

Dry heat (hot air oven)

160o/2hrs Materials that must remain dry

Treatment

Temp Effectiveness

Dry Heat (hot air oven)

170o/1hr

Same as above NB: Increasing temp by 10o shortens sterilizing time 50%


Pasteurization (batch method)

63o/30mins Kills most vegetative bacterial cells including pathogens, i.e., streptococci, staphylococci & Mycobacterium tuberculosis

Pasteurization (flash method)

72o/15secs Similar to batch methodFor milk conducive to industry fewer undesirable effects on quality & taste

Treatment

Temp Effectiveness


Sterilants: Chemical agent used in chemical sterilization

Disinfection: killing, inhibition or removal of microbes that may cause disease

Disinfectants: agents (chemical) used in disinfection only on inanimate objects

Sanitization: related to disinfection. Microbial popln reduced to levels considered safe by public health standards Antisepsis: prevention of infection or sepsis

Antiseptics: chemical agents applied to tissue to prevent infection by killing or inhibiting pathogen growth (not toxic)Germicide: kills pathogens & non pathogens (not endospores)Bactericide, Fungicide, Algicide or Viricide: disinfectant/antiseptic effective against specific microbial group

Some more words


Autoclave

to sterilize medium


Filtration apparatusto sterilize medium


Binary fission( 二分裂殖 ): 1 cell divides into 2 new cells

Growth rate: rate of cell to reproduction

Generation( 世代时 ): Time required for a complete fission cycle

i.e., 1 parent cell = 2 new daughter cells

1st Generation = 2 cells

2nd = 4 cells

3rd = 8 cells

How Bacterial Cells Divide


time

N

t = 0

“ Exponential growth”

time

Log

(N)

t = 0

“ Log growth”

2 x N DT = Doubling Time


Bacterium DT max

Bacillus stearothermophilus 8 min

E. coli 23 min

Caulobacter crescentus 90 min

Mycobacterium tuberculosis 6 hours


nt NN 20

0lglg

301.0

NN

tG

t

N0 is the number of cells at time zero. Nt is the number of cells at time t. n is the generations

G is the time needed to complete one generation.

Assume that cells are at the exponential growth stage. Then we can calculate cell numbers at ant time of the stage.


Growth stage in a batch culture

stationary

time

Log

(N)

lag

log

inoculate

decline


LAG PHASE 1. Newly inoculated cells, adjustment (can be diauxic) 2. NO cell division taking place 3. Population is sparse or dilute

LOG PHASE

1. Population growth at geometric/logarithmic rate 2. Cells reach maximum rate of cell division (while nutrients and environment are favorable)

STATIONARY PHASE

1. Population reaches maximum numbers, rate of cell inhibition (death) = Rate of multiplication

DEATH PHASE

1. Decline in growth rate (reverse Log phase) 2. Death in geometric fashion


culture

Continuous cultures:

Mediaflow

Outflow

Cells in continuous culture vassal can be kept at log phase.


The elemental composition of biomass is surprisingly constant across the variety of commercially utilised strains of bacteria and fungi.

For Example: C/N mole ratio

Escherichia coli CH1.77O0.49N0.24 ?

Saccharomyces cerevisiae CH1.83O0.56N0.17 ?

Pseudomonas C12B CH2.00O0.52N0.23 ?

Average CH1.79O0.50N0.20 ?

This average elemental composition can be utilised if no other value is available. However, values for many other organisms are available in literature.

The elemental composition of biomass


The end

chapter 3

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