Transcript Shixue Yin (Prof Dr)

Shixue Yin (Prof Dr)
CESE, Yangzhou University
Chapter 3
Microbial Growth
Shixue Yin (Prof Dr)
CESE, Yangzhou University
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).
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Growth requirements
Physical
1 Temperature
2 pH
3 Osmotic pressure (water)
4 Light
Chemical
1 Carbon
5 Inorganic element
2 Nitrogen
6 Oxygen
3 Sulfur
7 growth factors
4 Phosphorus
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Physical Requirements for
Growth: 1 temperature
• Temperature
–Minimum growth
temperature
–Optimum growth
temperature
–Maximum growth
temperature
Shixue Yin (Prof Dr)
CESE, Yangzhou University
专性嗜冷 兼性嗜冷
中温型
Arctic/antarctic mammal/soils
compost
Sea/refrige
嗜热
极端嗜热
Spring/sea volcano
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Question:
Is it always safe if foods are
stored in refrigerator for
long time?
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Figure 6.2
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Physical Requirements for
Growth: 2 pH
• 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
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Physical 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
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Plasmolysis
Cell in normal osmotic
pressure environment
Cell in hypertonic
environment
Figure 6.4
Shixue Yin (Prof Dr)
CESE, Yangzhou University
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)
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Chemical Requirements for
Growth: 1 carbon
• 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
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Chemical Requirements for
Growth:
1. Nitrogen
A.
B.
C.
D.
In amino acids, proteins
Most bacteria decompose proteins
Some bacteria use NH4+ or NO3
A few bacteria use N2 in nitrogen fixation
2. Sulfur
A. 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
Shixue Yin (Prof Dr)
CESE, Yangzhou University
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
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Chemical Requirements for
Growth:
5. Oxygen (O2)
Obligate Facultative Obligate
aerobes
anaerobes anaerobes
专性需氧菌 兼性厌氧菌 专性厌氧菌
O2 is
necessary
Grow
better if O2
O2 is toxic
Aerotolerant
anaerobes
微耐氧菌
O2 is not
necessary but
tolerable
Microaerophiles
微需氧菌
O2 is needed
at low partial
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Why O2 is toxic to some bacteria
• 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)
Some
bacteria
do not
have
these
enzymes
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Chemical Requirements for
Growth:
6. Organic Growth Factors
A. Organic compounds obtained from the
environment
B. Vitamins, amino acids, purines (嘌呤),
pyrimidines (嘧啶)
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Culture Media
• Culture Medium: Nutrients
prepared for microbial growth
• Sterile: No living microbes
• Inoculum: Introduction of microbes
into medium
• Culture: Microbes growing in/on
culture medium
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Agar
• 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
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Culture Media
• 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)
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Examples of Culture Media
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Anaerobic Culture Methods
• 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
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Anaerobic
Culture
Methods
• Anaerobic
jar
钯
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Anaerobic
Culture
Methods
• Anaerobic
chamber
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Some bacteria require high CO2
• Candle jar
O2-packet
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Selective
Media
• Enhance the
growth of
certain wanted
organisms but
suppress
unwanted
microbes.
Differential
Media
• Make it easy to
distinguish colonies
of different
microbes.
Figure 6.9b, c
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Enrichment 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
Shixue Yin (Prof Dr)
CESE, Yangzhou University
All-purposed (rich) medium
•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.
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Minimal Medium
•A Minimal medium supplies only the
minimal nutritional requirements of a
particular organism.
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Summary of Commonly-Used Constituents of Media
•Substance
•AGAR
Function
Composition
Solidifying agent Impure polysaccharide
Source
marine algae
•PEPTONES
nutrient
Animal/Plant proteins
cow,soy
•EXTRACTS
nutrient
Animal/Bacteria paste
cow,yeast
•BODY FLUIDS hormones
•BUFFERS
pH
animals
K2PO4;NaHPO3;CaCO3
•REDUCTANTS e- source
•SELECTIVES
bacteriostat
•INDICATORS
pH
•WATERhydration
Blood
thioglycolate
-
Antibiotics, sodium azide varies
bromothymol blue,phenol red -
H2O (DI & tap)
-
Shixue Yin (Prof Dr)
CESE, Yangzhou University
A few words
• 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 colonyforming unit (CFU)
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Streak Plate (平板划线)
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Medium sterilization
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Sterilization: all living cells, viable spores,
viruses are killed or removed from object
or habitat though:
Irradiation: destroys/distorts nucleic acids X-rays
microwaves. UV commonly used on object surfaces
&
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: formaldehyde, glutaraldehyde, ethylene oxide. Toxic
chemicals (require gas chamber) used for large items
Heat: important, widely used. Endospores (theromoduric)
destruction guarantees sterility
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Treatment
Incineration
Temp
>500o
Effectiveness
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
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Treatment
Temp
Effectiveness
Autoclave/pr 121o/15mins
essure cooker @ 15lbs
(steam under pressure
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
Dry Heat
(hot air oven)
170o/1hr
Same as above
NB: Increasing temp by 10o
shortens sterilizing time 50%
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Treatment
Pasteurization
(batch
method)
Temp
63o/30mins
Effectiveness
Kills most vegetative bacterial
cells including pathogens, i.e.,
streptococci, staphylococci &
Mycobacterium tuberculosis
Pasteurization 72o/15secs
(flash method)
Similar to batch method
For milk conducive to industry
fewer undesirable effects on
quality & taste
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Some more words
Sterilants: Chemical agent used in chemical sterilization
Disinfectants: agents (chemical) used in disinfection only on
inanimate objects
Disinfection: killing, inhibition or removal of microbes
that may cause disease
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:
effective against specific microbial group
disinfectant/antiseptic
Shixue Yin (Prof Dr)
CESE, Yangzhou University
to
sterilize
medium
Autoclave
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Filtration
apparatus
to
sterilize
medium
Shixue Yin (Prof Dr)
CESE, Yangzhou University
How Bacterial Cells Divide
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
Shixue Yin (Prof Dr)
CESE, Yangzhou University
N
“Exponential
growth”
time
t=0
Log(N)
“Log growth”
2xN
DT = Doubling Time
time
t=0
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Bacterium
DT max
Bacillus stearothermophilus
8 min
E. coli
23 min
Caulobacter crescentus
90 min
Mycobacterium tuberculosis
6 hours
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Assume that cells are at the exponential
growth stage. Then we can calculate cell
numbers at ant time of the stage.
Nt  N0 2
n
0.301t
G
lg N t  lg N 0
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.
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Growth stage in a batch culture
Log(N)
stationary
inoculate
decline
log
lag
time
Shixue Yin (Prof Dr)
CESE, Yangzhou University
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.
2.
Decline in growth rate (reverse Log phase)
Death in geometric fashion
Shixue Yin (Prof Dr)
CESE, Yangzhou University
Continuous cultures:
Media
flow
culture
Out
flow
Cells in continuous
culture vassal can
be kept at log
phase.
Shixue Yin (Prof Dr)
CESE, Yangzhou University
The elemental composition of biomass
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.
Shixue Yin (Prof Dr)
CESE, Yangzhou University
The end