Chapter 7 Microbial Growth
How to cultivate microorganisms?
How microorganisms grow?
What factors affect the microbial growth?
(Requirements for microbial growth)
How to control the microbial growth?
7.1 Measurement of microbial growth
7.1.1 Isolation of pure cultures
In natural habitats microorganisms usually grow in complex,
mixed populations containing severals pieces,This presents a
problem for microbiologist because a single type of microorganism
cannot be studied,
One needs a pure culture,a population of cells arising from a
single cell,to characterize a individual species,
Isolation of pure microorganisms,
spread-plate method
Spread culture method by dilution
Streak plate method
7.1.2 Culture Methods of Microorganisms
Be divided into two major categories depending on the
requirement of oxygen,
aerobic cultivation
anaerobic cultivation
Be divided into two major categories depending on the
physical peculiarity of culture medium,
liquid cultivation
solid cultivation
1) Solid aerobic cultivating methods
Solid cultivation in the test tube
and petri dish
7.1.3 Aerobic cultivating methods
Solid culture of mushroom
2） Aerobic liquid cultivating methods
Shaking incubator
Small-scale automatic fermentor for lab use
Anaerobic cultivation
Anaerobic chamber
Industrial fermentation equipment
Measurement of microbial growth
--Measure of cell numbers
1,Direct count methods do not distinguish between living and dead cells,and may be
accomplished by direct microscopic observation on specially etched slides (such as
Petroff-Hausser chambers or hemacytometers) or by using electronic counters (such as
Coulter Counters,which count microorganisms as they flow through a small hole or
orifice)
2,Viable cell counts involve plating diluted samples (using a pour plate or spread plate)
onto suitable growth media and monitoring colony formation; this type of method counts
only those cells that are reproductively active; because it is not possible to be certain that
each colony arose from a single cell,results are usually expressed as colony forming units
(CFU)
3,Microbial numbers are frequently determined from counts of colonies growing on
membrane filters having pores small enough to trap bacteria
--Measure of cell mass
may be used to approximate the number of microorganisms if a suitable parameter
proportional to the number of microorganisms present is used (suitable parameters may
be dry weight,light scattering in liquid solutions,or biochemical determinations of
specific cellular constituents such as protein,DNA,or ATP)
Measurement of cell numbers
1)Petroff-Hausser counting chamber
2) Filtration
3） Viable count using
serial dilutions of the
sample
4) Colonies count by membrane filtration
Measure of cell mass
1,Fresh weight
2,Dry Weight
3.Metabolic Activity
4,Turbidity
Turbidity
7.2 Growth
Microbial growth is at the level of
both the individual and the population
Individual growth = Increase in size and/or mass of an organism
with time
Population growth= increase in numbers of micro-organisms in
microbial populations
the knowledge of these two aspects can lead to the understanding of
the rules of microbial growth
?Unicellular microorganism- process the
increase in the cell size and mass then
divides into two separate progeny cells
?Multicellular microorgnism- process the
increase in the cell size and numbers
Individual growth
Bacterial growth
Rapidly growing- chromosome
is replicated before cell division
Slowly growing-chromosome is
replicated one before division
Ori
Figure 4-1
2,the cycle of reproduction,G （ gap phase） ),C （ chromosome
replication phase),D （ division phase）
3,control of the cell cycle in Escherichia coli
Initiation of DNA replication,initiation mass reached
Initiation of cell division,threshold length reached
4,Synthesis of cell wall
Figure 4-2 synthesis of cell wall
Yeast cell growth
1,Binary fission
Figure 4-3 Binary fission
2,budding
Figure 4-4 budding
Apical growth of Filamentous fungi
Figure 4-6
Figure 4-5 Saprolegnia ferax
0:00
1:00
Because microorganism are so small,
the growth of individual microorganisms
rarely studied,instead,when we
discuss microbial growth we inevitably
refer to microbial populations
Population growth?
Growth of unicellular microbial populations
unicellular microbes include
procaryotic bacteria and eucaryotic
yeast,The growth of the populations is
the increase in the number of cells,The
rate of microbial populations is the
increase in number and in cell mass per unit time,
1,A characteristic growth pattern –exponential phase
Logarithm of the
number Of bacteria is
related to time
0
30
60
90
120
150
0 30 60 90 120 150 180 210
培养时间
细胞数量
0.000
0.500
1.000
1.500
2.000
2.500
细胞数目对数
培养时间
细胞数量
细胞数目对数
Number
of
bacteria
Logarithm of the number of
bacteria
Time
Fomula
Nt= N0× 2n
lgNt=lgN0+nlg2
n = lgNt-lgN0 = lgNt-lgN0
lg2 0.301
N Number of generations
Nt Final Concentration of Cells
No Original concentration of cells
.301 Conversion Factor to Convert Log2 to Log10
Generation Time,The Time Required for a Cell to Divide
2,growth curve Phases of Microbial Growth
1） Lag Phase
During this period,the cell increases its size
It will synthesis new protoplasm,enzymes,coenzymes etc
The bacterium will adjust the physiological environment
The cells are metabolically active
At the end of lag phase,each cell will start reproduction
Factors
culture age
inoculation size
media composition
2） exponential phase
-All the cells are uniform chemical composition,
metabolic activity and physiological characters
-At this time,the generation time will be of constant
-The cells will be sensitive to chemical inhibitors
-This is the ideal stage to study the metabolic activity of
the bacterium
A number of factors can influence the growth rate of cells,
? The physical growth conditions
? The nutritional growth conditions
? The type of organism
? temperature
Practical significence
? good material for cellular and physiological research
? host for phage
? the best culture age
? determine the gram stain characteristics
3） Stationary Phase
-death rate = rate of reproduction
-A time of physiological change
-Gram-positive bacteria may begin to become variable in
the gram stain as the integrity of peptidoglycan comples
deteriorates
Population size remains constant?
-key nutrients are depleted
-toxic metabolic byproducts accumulate
-encounter environmental stress,
such as oxygen,pH
Practical significance
? optical harvest time for products
? second metabolisms and metabolic
products
? lead to the establishment of
continuous fermentation and
improvement of bio-technology
4） Death or decline phase
-Death rate > rate of reproduction
-The cell lysis occur during this stage
Related factors and practical significances
?Genetic characteristics
?Relating to the formation of endospores
?Nutrients and toxic metabolic byproducts
0
20
40
60
80
100
0 4 8 12 16 20 24 28 32
Cultivation time (h)
N
ic
ot
in
c
ac
i
d
an
d
6-
hy
dr
ox
yn
i
co
ti
ni
c
ac
i
d
(m
M)
0
1
2
3
4
5
OD
610nm
Fig.1,A time course of induced cultivation of Comamonas testosteroni
JA1,The cultivation was conducted in a 250-mL flask containing
50mL of medium I with 1 % (w/v) of nicotinic acid,(△ ),cell growth
(OD610nm); (■ ),6-hydroxynicotinic acid; and (□ ),nicotinic acid
were assayed with time,
Figure Liquid culture of filament fungus
Continuous culture
Batch and continuous culture
batch culture, A suspension culture in which cells grow in a finite
volume of liquid nutrient medium and follow a sigmoid pattern of
growth,All cells are harvested at the same time
Continuous culture,A suspension culture continuously supplied
with nutrients by the inflow of fresh medium,The culture volume is
normally constant,a process in which cells or micro-organisms are
maintained in culture in the exponential growth phase by the
continuous addition of fresh medium that is exactly balanced by the
removal of cell suspension from the bioreactor,
Continuous culture
chemostat turbidostat
Synchronous growth and culture
Synchronous growth of bacteria, All the cells of a
bacterial culture a same stage of their growth
cycle is referred as synchronous growth
synchronous culture,A culture in which the
microbial cell is synchronized for the majority of
the cells present,
Synchronous non-Synchronous Growth
Microbial growth in natural enviroments
Natural enviroment,
-Complex
-Constantly changing
Microbes’ response,
-changes in the morphology
-synthesis of special structures and enzymes
Growth limitation by environmental factors
1,Liebig's Law of the Minimum, The growth and
reproduction of an microorganism is dependent on
the nutrient substance that is availble
in minimum quantity
2,Shelford’s law of tolerances, For an organism to
succeed in a given environment,each of a complex
set of conditions must remain within the tolerance
range of that organism and if any one condition
exceeds the minimum or maximum tolerance of that
organism,it will fail to thrive
these two rules also can be applied to the cultured microorganisms
Nonculturable microbes
A viable microorganism- one that is able to grow actively,
resulting in the formation of a colony or visible turbidity in liquid
medium
Nonculturable microorganism- John R,Postgate first
defined it as that those viable microorgansim of sensitive to
secondary stresses are not able to grow normal laboratory media
-Postgate Microviability Assay
-sensitive microscope
-isotopic procedure
-molecular techniques
Some studies showed when bacteria have lost their ability to
grow on conventional laboratory media using standard culture
techniques,they still might be able to play a role in infectious
diseases,
Practical sense
１,diversity
２,new resource
3,knowledge of new pathogen and its mechanisms
Quorum sensing and Microbial population
More recently,it has become clear that many
bacteria can communicate with one another and
behave cooperatively through quorum sensing or
autoinduction
Quorum sensing –bacteria monitor their own population density
through sensing the level of signal molecules,and some times called
autoinducers because they can stimulate the cell that releases them,
The concentration of these signal molecules increases along with the
bacteria population until is rises to a specific threshold and
signals the bacteria that the population density has reached a
critical level or quorum,The bacteria then begin expressing sets of
quorum-dependent genes
Important function of quorum sensing
If extracellular enzymes were released by only a few bacteria,
they would diffuse away and be rendered ineffective because of
dilution,With control by quorum sensing,the bacteria reach a high
population density before they release enzymes,and as a consequence
enzyme levels are concentrated enough to have significant effects,
If a pathogen can reach high levels at a particular site before
producing virulence factors and escaping into surrounding host
tissues,it has a much better chance of counteracting hose defenses
and successfully spreading throughout the host’s body,
It is very important in many bacteria that establish symbiotic or
parasitic relationship with hosts,
The influence of
environmental factors on growth
Temperature and growth
pH
1,Acidophiles grow best between pH 0 and 5.5
2,Neutrophiles grow best between pH 5.5 and 8.0
3,Alkalophiles grow best between pH 8.5 and 11.5
4,Extreme alkalophiles grow best at pH 10.0 or higher
5,Despite wide variations in habitat pH,the internal pH of most
microorganisms is maintained near neutrality either by
proton/ion exchange or by internal buffering
Oxygen
-is lethal to some organisms,
All organisms produce superoxide ( O2-)
Superoxide is toxic to cells (steals electrons)
Superoxide must be neutralized
O2+e? O2 (O2), -
Superoxide dismutase
?O2- + O2- + 2 H+ -------> H2O2 + O2
Catalase
?2 H2O2 --------> 2 H2O + O2
According to the effect of the controlling
sterilization,the process that can destroy or remove all viable
microorganisms,including viruses in a given area；
disinfection,the ways that can kill,remove or reduce the
pathogenic organisms from an object ；
antisepsis,chemicals applied to body surfaces to destroy or
inhibit vegetative pathogens。
According to the principles and methods of the controlling
Physical methods for controlling
Chemical methods for controlling
7.4 controlling microorganisms
Factors that influence the rate at which microbes are killed by antimicrobial agents,
The total original microbial
Relative resistance
Length of
exposure to the
agent
Action of the agent
7.4.1 Physical methods for controlling
microorganisms
(1) Temperature
? dry oven, exposure to 1500C～ 1700C for 1 to 2 hours,
substances appropriate are glassware,metallic instruments,
powders and oils that steam does not penetrate well。
advantages, can keep the dryness of the substances
?Incineration (flame),substances appropriate are metallic
instruments used as inoculation,polluted or used materials。
1,dry-heat
Moist –heat
makes use of the
steam。
2.moist-heat
The effect of the
steam is better
than that of the
dry-heat on the
same
temperature,
WHY？
① the katogene of hot steam to cell component is stronger
② the penetrating power of hot stream is stronger than hot air
③ latent hot of stream is strong,a great quantity of heat is
released when gas changes into liquid,therefore the
temperature of sterilization is increased rapidly,
?Boiling water,exposure to the water which is 1000C for more
than 15min。
autoclave,applied widely,Put the objects into sterilizing chamber
where there is a little water,As steam flows from the steam jacket
into the sterilizing chamber,the cool air is forced out and a special
valve increases the pressure gradually and the temperature rises to
121 and the super-heated water molecules rapidly conduct heat into
the microorganisms,The time for destruction of the most resistant
bacterial spore is about 15min to 20min,The lower temperature --
1150C can also be utilized but should be kept for 35min,The
autoclave occupies an important position in the control of
microorganisms in hospitals,laboratories and fermentation plant,
2,moist heat
boiling water,autoclave,intermittent sterilization,
pasteurization
Note that it is not the pressure of the autoclave that kills the
microorganisms,but the high temperature that can be achieved
when steam is placed under pressure,As we shall know,increasing
pressure can increase the boiling point of water,so that the
temperature of steam can be increased,
?pasteurization,a technique in which heat is applied to
liquids to kill potential agents of infection such as Coxiella
and Mycobacterium and spoilage,while at the same time
retaining the liquid’s flavor and food value,Usually at the
temperature 630C for 30min or 720C for 15min。 Such as
milk,beer,ratafee,sauce can be applied with it,
? intermittent sterilization,repeat the exposure to free-flowing steam.
（ 2） cold treatment
The principal benefit of cold treatment is to slow growth of
cultures and microbes in food during processing and storage by
slowing the rate of enzymes’ action,
1,chilling
Keep the fresh food at 5℃ ( usually the temperature of
refrigerator’s safe) from putrescence,However,it can be kept
only for days because some psychrophiles grow very slowly even
at low temperatures,
2,freezing
In families or food industries the food is frozen at -10 ℃ ~ -
20℃ at which the most microorganisms can not grow,So it can
be kept longer by freezing than by chilling,
(3)drying and hypso-osmotic pressure
Since microorganisms do not grow at low water activities,microbial growth
can be controlled by drying or controlling osmotic pressure,
1.Dryness
Drying is to dehydrate the materials,Drying can seldom kill the
microorganisms but can inhibit the growth of them,
Sensitivity to dryness of different microorganisms,
Micrococcus gonococcus sensitive to dryness die after a few hours
Bacillus tuberculosis resist dryness alive at 100℃ for 20min
chain coccus resist dryness be preserved for years without losing
pathogenicity
cystospore strongly strand dryness remain viable for lengthy periods in
arid conditions
2,osmotic pressure
Increasing osmotic pressure is another way to inhibit the growth of
microorganisms by lowering water activity,Foods preserved by addition of
sugar are mainly fruits,Salted products are primarily meats and fish,
（ 4） radiation
Four types of radiation,UV radiation,ionizing radiation,strong
visible radiation in a given condition,microwave,which can be
used as control the growth of microorganisms and preserve the food,
1.UV radiation
? It is most lethal in ranges of wavelength from 200 to 300nm,
? Mechanism of its antibacterial,proteins (app 280nm) and nucleic acids
(app 260nm) denature by absorbing them,Lethal effects appear to be chemical
changes in organelles and the production of toxic photochemical products—
free radicals,which binding to DNA,RNA and proteins to interfere with
essential cell processes,
? Because UV radiation passes readily through air,slightly through liquids,
and only poorly through solids such as glasses,clothes and paper,the object to
be disinfected not sterile must be directly exposed to it for full effect,
? Mostly applied to the disinfection of the objects’ surface and room air,Some
special devices of UV radiation can be used to disinfect fluid such as drinking
water instead of heating and bleaching powder,
2,ionizing radiation
? Includes Gamma Rays and X Rays,When X rays or gamma
rays bombard a cell,it ejects orbital electrons from an atom,it
causes ions to form,This is the effect of ionizing radiation,
Ionizing radiation can also form H2O2 to denature the proteins
even to kill the cell,
? Ionizing radiation is mostly applied to the sterilization of plastic
material,medical treatment facility,pharmaceutical product and
foods which are sensitive to other sterilizations,
3.Strong visible radiation
The strong visible radiation ranging in wavelength from 400 to
700nm also has direct effect of sterilization,they can oxidize the
intracellular photaesthesia,such as lactochrome and porphyrin
ring (the composition of oxidase),
The bacteria culture in the lab should be avoided exposing to
strong visible radiation,Caesar red and Methylene Blue can
absorb the energy of strong visible radiation and oxidize the
proteins and nucleic acids,therefore they are often combined as
sterilization to bacteria and virus,
4.microwaves
Microwaves are infrared rays,Microwaves can not kill the
microorganisms directly,while they cause the objects to become
hot,which kill the microorganisms,However,it always happens
that microorganisms escape heating of microwaves because of
uneven matter,
(V)Filtration Removes microbes from heat-sensitive liquids and
circulating air using a filter,As fluid passes through,
the organisms are trapped in the pores of the filtering
material,
（ a）注射式微孔滤器 （ b）小型过滤装置 （ c）层流生物安全柜
图 6—16 常用膜滤器
Chemical methods mainly use a serious of chemical
agents to control the growth of microorganisms,
antimicrobial chemical agents
different physical condition,
liquid,gasiform and solid
different effect and influence on human being or
animals,
antiseptics,antiseptics,chemotherapeutic agent
7.4.2 chemical methods for controlling
microorganisms
7.4.2.1 Disinfectant and antiseptics
disinfectant are chemicals that kill microorganisms but
also do harm to the humans and are used on inanimate objects
such as the surface of objects,equipments and dejectures,
antiseptics are chemical agents that kill or inhibit growth
of microorganisms and that are sufficiently nontoxic to be applied
to living tissues,They can prevent the surface of bodies,such as
skins,mucosa and wounds etc from infecting,They are also used
to antisepticize foods,drinking and drug products,
But now,the distinction of disinfectant and antiseptics is
not obvious strictly,For example,high concentration alcohol
phenyl （ 3%-5%） is used to disinfect the surface of utensil，
while low concentration （ 0.5% ） is used to antisepticize
biological products,
The ideal chemical disinfectant and antiseptics should take
a quick and good effect but has little tissue damage with strong
penetrativity and little corrosion,They should be made easily
and be stable with cheapness and without unpleasant smell,
7.4.2.2 Chemical Agents
Chemical Agents it must be able to kill certain microbes
especially and selectivly,but it should also be nontoxic to
animals or humans,it could be used to therapy disease caused
by microbes,
1,Chemotherapeutic Agents
Agents which are similar to the necessary metabolite of cell in
chemical structure,and can interfere normal metabolism
activity,
Sulfanilamide was the first of a group of chemotherapeutic
agents known as sulfonamides,One of the components of folic
acid is para-aminobenzoic acid （ PABA ） is similar to
Sulfanilamide in chemical structure,Folic acid is a coenzyme,it
plays an important role in the synthesis of amino acid,vitamin,
nucleic acid and protein,Certain bacteria must synthesize folic
acid for use in nucleic acid production,
Why Sulfanilamide is nontoxic to human cells?
Humans cannot synthesize their own folic acid because of the
lack of enzymes -- that can synthesize folic acid from PABA,
therefore they cannot use PABA to synthesize folic acid,and
must have it supplied to them,either in foods or in the pure
form in vitamin capsules,
2,Antibiotics
Antibiotics is composed by two Greece words,anti means
― antagonize,, bios means ―being‖,join together，
antibiotics means ability to kill beings,that is,antibiotics is a
group of matters that can kill microorganisms,
Antibiotics are chemical substances produced by certain
microorganisms which are active against other
microorganisms,Antibiotics constitute a special class of
chemotherapeutic agents,distinguished most importantly by
the fact that they are natural products (products of microbial
activity),They constitute one of the most important classes of
substances produced by larger-scale microbial processes,
Antibiotics was first noticed by an
English scientist A.Fieming in 1928,and
used as chemotherapeutic agents in
1940’s,
Penicilin inhibit the growth
Micrococcus luteus
Antimicrobial spectrum of antibiotics,The range of objects
effected by antibiotics,Antibiotics which have effect on many
classes of bacteria were called broad spectrum antibiotics,e.g,
acheomycin and terramycin,have effect on both gram-positive and
gram-negative bacteria; those which only have effect on a few
bacteria were called narrow spectrum antibiotics,e.g,penicillin
only have effect on gram-positive bacteria,
antibiotics titer,a measuring unity of the quantity
active component of microamount antibiotics,
Some use the weight of correspond biological activity
as unit,e.g,1μg=1Unit(U),phytomycin muriaficus
was expressed by this way,
Some use the actual weight of biological activity of
pure antibiotic as 1Unit,e.g,penicillin unit was first
measured by its ability to use the least quantity but
totally prevent Staphylococcus aureus in 50ml bouillon
culture-medium as 1Unit,After the purification of
penicillin,1Unit was definited to be 0.5988μg
benzylpenicillin sodium, so 0.5988μg benzylpenicillin
sodium was used as 1Unit penicillin,
Antibiotic mechanism of
action
（ 3） Drug resistance of microorganisms
Six mechanisms of drug resistance,
① change structure so that some drug cannot affect,In the case of penicillin,by
converting to a cell-wall-deficient form (L form) that penicillin cannot affect,
② decrease in cell permeability and uptake of the drug,Streptomyces venezuelae
prevents cyclomycin from entering cell by changing permeability of membrane,
③ Many bacteria possess multidrug resistant pumps that actively transport
drugs and other chemicals out of cells,Some resistant bacteria like E,coli and
Staphylococcus aureus can pump proton in and drug out of cell by proton/drug
exchanger in membrane,
④ synthesis of enzymes that inactivate the drug,For example,some
Staphylococcus aureus produce lactamase,which can break thelactam ring and lose
bacteriostatic action;
⑤ change in the number or affinity of the drug receptor sites,Because most
drugs act on a specific target such as protein,RNA,DNA,or membrane
structure,microbes can circumvent drugs by altering the nature of this target,
Phytomycin resistance is associated with an alteration on the 30S ribosomal binding
site,
⑥ modification of an essential metabolic pathway,Some strains resistant to
sulfonic acid alter the nature of dihydrofolate synthetase and synthesize an enzyme
which is insensitive to sulfonic acid,
Inherent resistance to antibiotic
Spread of resistance factors
References
Prescott LM,Harley JP,and Klein DA.,Microbiology (5th ed.),Higher education
press and McGraw-Hill Companies,Inc.2002
Michael TM,John MM,Jack P,Brock.,Biology of Micoorganisms,International
edition,Pearson Education,Inc.2003
Talaro K,P,Foundations in Microbiology (Fifth Edition),Higher education press
and McGraw-Hill Companies,Inc.2005
Zhou De-qing,Microbiology textbook,second edition,high education publishing
house,2002
How to cultivate microorganisms?
How microorganisms grow?
What factors affect the microbial growth?
(Requirements for microbial growth)
How to control the microbial growth?
7.1 Measurement of microbial growth
7.1.1 Isolation of pure cultures
In natural habitats microorganisms usually grow in complex,
mixed populations containing severals pieces,This presents a
problem for microbiologist because a single type of microorganism
cannot be studied,
One needs a pure culture,a population of cells arising from a
single cell,to characterize a individual species,
Isolation of pure microorganisms,
spread-plate method
Spread culture method by dilution
Streak plate method
7.1.2 Culture Methods of Microorganisms
Be divided into two major categories depending on the
requirement of oxygen,
aerobic cultivation
anaerobic cultivation
Be divided into two major categories depending on the
physical peculiarity of culture medium,
liquid cultivation
solid cultivation
1) Solid aerobic cultivating methods
Solid cultivation in the test tube
and petri dish
7.1.3 Aerobic cultivating methods
Solid culture of mushroom
2） Aerobic liquid cultivating methods
Shaking incubator
Small-scale automatic fermentor for lab use
Anaerobic cultivation
Anaerobic chamber
Industrial fermentation equipment
Measurement of microbial growth
--Measure of cell numbers
1,Direct count methods do not distinguish between living and dead cells,and may be
accomplished by direct microscopic observation on specially etched slides (such as
Petroff-Hausser chambers or hemacytometers) or by using electronic counters (such as
Coulter Counters,which count microorganisms as they flow through a small hole or
orifice)
2,Viable cell counts involve plating diluted samples (using a pour plate or spread plate)
onto suitable growth media and monitoring colony formation; this type of method counts
only those cells that are reproductively active; because it is not possible to be certain that
each colony arose from a single cell,results are usually expressed as colony forming units
(CFU)
3,Microbial numbers are frequently determined from counts of colonies growing on
membrane filters having pores small enough to trap bacteria
--Measure of cell mass
may be used to approximate the number of microorganisms if a suitable parameter
proportional to the number of microorganisms present is used (suitable parameters may
be dry weight,light scattering in liquid solutions,or biochemical determinations of
specific cellular constituents such as protein,DNA,or ATP)
Measurement of cell numbers
1)Petroff-Hausser counting chamber
2) Filtration
3） Viable count using
serial dilutions of the
sample
4) Colonies count by membrane filtration
Measure of cell mass
1,Fresh weight
2,Dry Weight
3.Metabolic Activity
4,Turbidity
Turbidity
7.2 Growth
Microbial growth is at the level of
both the individual and the population
Individual growth = Increase in size and/or mass of an organism
with time
Population growth= increase in numbers of micro-organisms in
microbial populations
the knowledge of these two aspects can lead to the understanding of
the rules of microbial growth
?Unicellular microorganism- process the
increase in the cell size and mass then
divides into two separate progeny cells
?Multicellular microorgnism- process the
increase in the cell size and numbers
Individual growth
Bacterial growth
Rapidly growing- chromosome
is replicated before cell division
Slowly growing-chromosome is
replicated one before division
Ori
Figure 4-1
2,the cycle of reproduction,G （ gap phase） ),C （ chromosome
replication phase),D （ division phase）
3,control of the cell cycle in Escherichia coli
Initiation of DNA replication,initiation mass reached
Initiation of cell division,threshold length reached
4,Synthesis of cell wall
Figure 4-2 synthesis of cell wall
Yeast cell growth
1,Binary fission
Figure 4-3 Binary fission
2,budding
Figure 4-4 budding
Apical growth of Filamentous fungi
Figure 4-6
Figure 4-5 Saprolegnia ferax
0:00
1:00
Because microorganism are so small,
the growth of individual microorganisms
rarely studied,instead,when we
discuss microbial growth we inevitably
refer to microbial populations
Population growth?
Growth of unicellular microbial populations
unicellular microbes include
procaryotic bacteria and eucaryotic
yeast,The growth of the populations is
the increase in the number of cells,The
rate of microbial populations is the
increase in number and in cell mass per unit time,
1,A characteristic growth pattern –exponential phase
Logarithm of the
number Of bacteria is
related to time
0
30
60
90
120
150
0 30 60 90 120 150 180 210
培养时间
细胞数量
0.000
0.500
1.000
1.500
2.000
2.500
细胞数目对数
培养时间
细胞数量
细胞数目对数
Number
of
bacteria
Logarithm of the number of
bacteria
Time
Fomula
Nt= N0× 2n
lgNt=lgN0+nlg2
n = lgNt-lgN0 = lgNt-lgN0
lg2 0.301
N Number of generations
Nt Final Concentration of Cells
No Original concentration of cells
.301 Conversion Factor to Convert Log2 to Log10
Generation Time,The Time Required for a Cell to Divide
2,growth curve Phases of Microbial Growth
1） Lag Phase
During this period,the cell increases its size
It will synthesis new protoplasm,enzymes,coenzymes etc
The bacterium will adjust the physiological environment
The cells are metabolically active
At the end of lag phase,each cell will start reproduction
Factors
culture age
inoculation size
media composition
2） exponential phase
-All the cells are uniform chemical composition,
metabolic activity and physiological characters
-At this time,the generation time will be of constant
-The cells will be sensitive to chemical inhibitors
-This is the ideal stage to study the metabolic activity of
the bacterium
A number of factors can influence the growth rate of cells,
? The physical growth conditions
? The nutritional growth conditions
? The type of organism
? temperature
Practical significence
? good material for cellular and physiological research
? host for phage
? the best culture age
? determine the gram stain characteristics
3） Stationary Phase
-death rate = rate of reproduction
-A time of physiological change
-Gram-positive bacteria may begin to become variable in
the gram stain as the integrity of peptidoglycan comples
deteriorates
Population size remains constant?
-key nutrients are depleted
-toxic metabolic byproducts accumulate
-encounter environmental stress,
such as oxygen,pH
Practical significance
? optical harvest time for products
? second metabolisms and metabolic
products
? lead to the establishment of
continuous fermentation and
improvement of bio-technology
4） Death or decline phase
-Death rate > rate of reproduction
-The cell lysis occur during this stage
Related factors and practical significances
?Genetic characteristics
?Relating to the formation of endospores
?Nutrients and toxic metabolic byproducts
0
20
40
60
80
100
0 4 8 12 16 20 24 28 32
Cultivation time (h)
N
ic
ot
in
c
ac
i
d
an
d
6-
hy
dr
ox
yn
i
co
ti
ni
c
ac
i
d
(m
M)
0
1
2
3
4
5
OD
610nm
Fig.1,A time course of induced cultivation of Comamonas testosteroni
JA1,The cultivation was conducted in a 250-mL flask containing
50mL of medium I with 1 % (w/v) of nicotinic acid,(△ ),cell growth
(OD610nm); (■ ),6-hydroxynicotinic acid; and (□ ),nicotinic acid
were assayed with time,
Figure Liquid culture of filament fungus
Continuous culture
Batch and continuous culture
batch culture, A suspension culture in which cells grow in a finite
volume of liquid nutrient medium and follow a sigmoid pattern of
growth,All cells are harvested at the same time
Continuous culture,A suspension culture continuously supplied
with nutrients by the inflow of fresh medium,The culture volume is
normally constant,a process in which cells or micro-organisms are
maintained in culture in the exponential growth phase by the
continuous addition of fresh medium that is exactly balanced by the
removal of cell suspension from the bioreactor,
Continuous culture
chemostat turbidostat
Synchronous growth and culture
Synchronous growth of bacteria, All the cells of a
bacterial culture a same stage of their growth
cycle is referred as synchronous growth
synchronous culture,A culture in which the
microbial cell is synchronized for the majority of
the cells present,
Synchronous non-Synchronous Growth
Microbial growth in natural enviroments
Natural enviroment,
-Complex
-Constantly changing
Microbes’ response,
-changes in the morphology
-synthesis of special structures and enzymes
Growth limitation by environmental factors
1,Liebig's Law of the Minimum, The growth and
reproduction of an microorganism is dependent on
the nutrient substance that is availble
in minimum quantity
2,Shelford’s law of tolerances, For an organism to
succeed in a given environment,each of a complex
set of conditions must remain within the tolerance
range of that organism and if any one condition
exceeds the minimum or maximum tolerance of that
organism,it will fail to thrive
these two rules also can be applied to the cultured microorganisms
Nonculturable microbes
A viable microorganism- one that is able to grow actively,
resulting in the formation of a colony or visible turbidity in liquid
medium
Nonculturable microorganism- John R,Postgate first
defined it as that those viable microorgansim of sensitive to
secondary stresses are not able to grow normal laboratory media
-Postgate Microviability Assay
-sensitive microscope
-isotopic procedure
-molecular techniques
Some studies showed when bacteria have lost their ability to
grow on conventional laboratory media using standard culture
techniques,they still might be able to play a role in infectious
diseases,
Practical sense
１,diversity
２,new resource
3,knowledge of new pathogen and its mechanisms
Quorum sensing and Microbial population
More recently,it has become clear that many
bacteria can communicate with one another and
behave cooperatively through quorum sensing or
autoinduction
Quorum sensing –bacteria monitor their own population density
through sensing the level of signal molecules,and some times called
autoinducers because they can stimulate the cell that releases them,
The concentration of these signal molecules increases along with the
bacteria population until is rises to a specific threshold and
signals the bacteria that the population density has reached a
critical level or quorum,The bacteria then begin expressing sets of
quorum-dependent genes
Important function of quorum sensing
If extracellular enzymes were released by only a few bacteria,
they would diffuse away and be rendered ineffective because of
dilution,With control by quorum sensing,the bacteria reach a high
population density before they release enzymes,and as a consequence
enzyme levels are concentrated enough to have significant effects,
If a pathogen can reach high levels at a particular site before
producing virulence factors and escaping into surrounding host
tissues,it has a much better chance of counteracting hose defenses
and successfully spreading throughout the host’s body,
It is very important in many bacteria that establish symbiotic or
parasitic relationship with hosts,
The influence of
environmental factors on growth
Temperature and growth
pH
1,Acidophiles grow best between pH 0 and 5.5
2,Neutrophiles grow best between pH 5.5 and 8.0
3,Alkalophiles grow best between pH 8.5 and 11.5
4,Extreme alkalophiles grow best at pH 10.0 or higher
5,Despite wide variations in habitat pH,the internal pH of most
microorganisms is maintained near neutrality either by
proton/ion exchange or by internal buffering
Oxygen
-is lethal to some organisms,
All organisms produce superoxide ( O2-)
Superoxide is toxic to cells (steals electrons)
Superoxide must be neutralized
O2+e? O2 (O2), -
Superoxide dismutase
?O2- + O2- + 2 H+ -------> H2O2 + O2
Catalase
?2 H2O2 --------> 2 H2O + O2
According to the effect of the controlling
sterilization,the process that can destroy or remove all viable
microorganisms,including viruses in a given area；
disinfection,the ways that can kill,remove or reduce the
pathogenic organisms from an object ；
antisepsis,chemicals applied to body surfaces to destroy or
inhibit vegetative pathogens。
According to the principles and methods of the controlling
Physical methods for controlling
Chemical methods for controlling
7.4 controlling microorganisms
Factors that influence the rate at which microbes are killed by antimicrobial agents,
The total original microbial
Relative resistance
Length of
exposure to the
agent
Action of the agent
7.4.1 Physical methods for controlling
microorganisms
(1) Temperature
? dry oven, exposure to 1500C～ 1700C for 1 to 2 hours,
substances appropriate are glassware,metallic instruments,
powders and oils that steam does not penetrate well。
advantages, can keep the dryness of the substances
?Incineration (flame),substances appropriate are metallic
instruments used as inoculation,polluted or used materials。
1,dry-heat
Moist –heat
makes use of the
steam。
2.moist-heat
The effect of the
steam is better
than that of the
dry-heat on the
same
temperature,
WHY？
① the katogene of hot steam to cell component is stronger
② the penetrating power of hot stream is stronger than hot air
③ latent hot of stream is strong,a great quantity of heat is
released when gas changes into liquid,therefore the
temperature of sterilization is increased rapidly,
?Boiling water,exposure to the water which is 1000C for more
than 15min。
autoclave,applied widely,Put the objects into sterilizing chamber
where there is a little water,As steam flows from the steam jacket
into the sterilizing chamber,the cool air is forced out and a special
valve increases the pressure gradually and the temperature rises to
121 and the super-heated water molecules rapidly conduct heat into
the microorganisms,The time for destruction of the most resistant
bacterial spore is about 15min to 20min,The lower temperature --
1150C can also be utilized but should be kept for 35min,The
autoclave occupies an important position in the control of
microorganisms in hospitals,laboratories and fermentation plant,
2,moist heat
boiling water,autoclave,intermittent sterilization,
pasteurization
Note that it is not the pressure of the autoclave that kills the
microorganisms,but the high temperature that can be achieved
when steam is placed under pressure,As we shall know,increasing
pressure can increase the boiling point of water,so that the
temperature of steam can be increased,
?pasteurization,a technique in which heat is applied to
liquids to kill potential agents of infection such as Coxiella
and Mycobacterium and spoilage,while at the same time
retaining the liquid’s flavor and food value,Usually at the
temperature 630C for 30min or 720C for 15min。 Such as
milk,beer,ratafee,sauce can be applied with it,
? intermittent sterilization,repeat the exposure to free-flowing steam.
（ 2） cold treatment
The principal benefit of cold treatment is to slow growth of
cultures and microbes in food during processing and storage by
slowing the rate of enzymes’ action,
1,chilling
Keep the fresh food at 5℃ ( usually the temperature of
refrigerator’s safe) from putrescence,However,it can be kept
only for days because some psychrophiles grow very slowly even
at low temperatures,
2,freezing
In families or food industries the food is frozen at -10 ℃ ~ -
20℃ at which the most microorganisms can not grow,So it can
be kept longer by freezing than by chilling,
(3)drying and hypso-osmotic pressure
Since microorganisms do not grow at low water activities,microbial growth
can be controlled by drying or controlling osmotic pressure,
1.Dryness
Drying is to dehydrate the materials,Drying can seldom kill the
microorganisms but can inhibit the growth of them,
Sensitivity to dryness of different microorganisms,
Micrococcus gonococcus sensitive to dryness die after a few hours
Bacillus tuberculosis resist dryness alive at 100℃ for 20min
chain coccus resist dryness be preserved for years without losing
pathogenicity
cystospore strongly strand dryness remain viable for lengthy periods in
arid conditions
2,osmotic pressure
Increasing osmotic pressure is another way to inhibit the growth of
microorganisms by lowering water activity,Foods preserved by addition of
sugar are mainly fruits,Salted products are primarily meats and fish,
（ 4） radiation
Four types of radiation,UV radiation,ionizing radiation,strong
visible radiation in a given condition,microwave,which can be
used as control the growth of microorganisms and preserve the food,
1.UV radiation
? It is most lethal in ranges of wavelength from 200 to 300nm,
? Mechanism of its antibacterial,proteins (app 280nm) and nucleic acids
(app 260nm) denature by absorbing them,Lethal effects appear to be chemical
changes in organelles and the production of toxic photochemical products—
free radicals,which binding to DNA,RNA and proteins to interfere with
essential cell processes,
? Because UV radiation passes readily through air,slightly through liquids,
and only poorly through solids such as glasses,clothes and paper,the object to
be disinfected not sterile must be directly exposed to it for full effect,
? Mostly applied to the disinfection of the objects’ surface and room air,Some
special devices of UV radiation can be used to disinfect fluid such as drinking
water instead of heating and bleaching powder,
2,ionizing radiation
? Includes Gamma Rays and X Rays,When X rays or gamma
rays bombard a cell,it ejects orbital electrons from an atom,it
causes ions to form,This is the effect of ionizing radiation,
Ionizing radiation can also form H2O2 to denature the proteins
even to kill the cell,
? Ionizing radiation is mostly applied to the sterilization of plastic
material,medical treatment facility,pharmaceutical product and
foods which are sensitive to other sterilizations,
3.Strong visible radiation
The strong visible radiation ranging in wavelength from 400 to
700nm also has direct effect of sterilization,they can oxidize the
intracellular photaesthesia,such as lactochrome and porphyrin
ring (the composition of oxidase),
The bacteria culture in the lab should be avoided exposing to
strong visible radiation,Caesar red and Methylene Blue can
absorb the energy of strong visible radiation and oxidize the
proteins and nucleic acids,therefore they are often combined as
sterilization to bacteria and virus,
4.microwaves
Microwaves are infrared rays,Microwaves can not kill the
microorganisms directly,while they cause the objects to become
hot,which kill the microorganisms,However,it always happens
that microorganisms escape heating of microwaves because of
uneven matter,
(V)Filtration Removes microbes from heat-sensitive liquids and
circulating air using a filter,As fluid passes through,
the organisms are trapped in the pores of the filtering
material,
（ a）注射式微孔滤器 （ b）小型过滤装置 （ c）层流生物安全柜
图 6—16 常用膜滤器
Chemical methods mainly use a serious of chemical
agents to control the growth of microorganisms,
antimicrobial chemical agents
different physical condition,
liquid,gasiform and solid
different effect and influence on human being or
animals,
antiseptics,antiseptics,chemotherapeutic agent
7.4.2 chemical methods for controlling
microorganisms
7.4.2.1 Disinfectant and antiseptics
disinfectant are chemicals that kill microorganisms but
also do harm to the humans and are used on inanimate objects
such as the surface of objects,equipments and dejectures,
antiseptics are chemical agents that kill or inhibit growth
of microorganisms and that are sufficiently nontoxic to be applied
to living tissues,They can prevent the surface of bodies,such as
skins,mucosa and wounds etc from infecting,They are also used
to antisepticize foods,drinking and drug products,
But now,the distinction of disinfectant and antiseptics is
not obvious strictly,For example,high concentration alcohol
phenyl （ 3%-5%） is used to disinfect the surface of utensil，
while low concentration （ 0.5% ） is used to antisepticize
biological products,
The ideal chemical disinfectant and antiseptics should take
a quick and good effect but has little tissue damage with strong
penetrativity and little corrosion,They should be made easily
and be stable with cheapness and without unpleasant smell,
7.4.2.2 Chemical Agents
Chemical Agents it must be able to kill certain microbes
especially and selectivly,but it should also be nontoxic to
animals or humans,it could be used to therapy disease caused
by microbes,
1,Chemotherapeutic Agents
Agents which are similar to the necessary metabolite of cell in
chemical structure,and can interfere normal metabolism
activity,
Sulfanilamide was the first of a group of chemotherapeutic
agents known as sulfonamides,One of the components of folic
acid is para-aminobenzoic acid （ PABA ） is similar to
Sulfanilamide in chemical structure,Folic acid is a coenzyme,it
plays an important role in the synthesis of amino acid,vitamin,
nucleic acid and protein,Certain bacteria must synthesize folic
acid for use in nucleic acid production,
Why Sulfanilamide is nontoxic to human cells?
Humans cannot synthesize their own folic acid because of the
lack of enzymes -- that can synthesize folic acid from PABA,
therefore they cannot use PABA to synthesize folic acid,and
must have it supplied to them,either in foods or in the pure
form in vitamin capsules,
2,Antibiotics
Antibiotics is composed by two Greece words,anti means
― antagonize,, bios means ―being‖,join together，
antibiotics means ability to kill beings,that is,antibiotics is a
group of matters that can kill microorganisms,
Antibiotics are chemical substances produced by certain
microorganisms which are active against other
microorganisms,Antibiotics constitute a special class of
chemotherapeutic agents,distinguished most importantly by
the fact that they are natural products (products of microbial
activity),They constitute one of the most important classes of
substances produced by larger-scale microbial processes,
Antibiotics was first noticed by an
English scientist A.Fieming in 1928,and
used as chemotherapeutic agents in
1940’s,
Penicilin inhibit the growth
Micrococcus luteus
Antimicrobial spectrum of antibiotics,The range of objects
effected by antibiotics,Antibiotics which have effect on many
classes of bacteria were called broad spectrum antibiotics,e.g,
acheomycin and terramycin,have effect on both gram-positive and
gram-negative bacteria; those which only have effect on a few
bacteria were called narrow spectrum antibiotics,e.g,penicillin
only have effect on gram-positive bacteria,
antibiotics titer,a measuring unity of the quantity
active component of microamount antibiotics,
Some use the weight of correspond biological activity
as unit,e.g,1μg=1Unit(U),phytomycin muriaficus
was expressed by this way,
Some use the actual weight of biological activity of
pure antibiotic as 1Unit,e.g,penicillin unit was first
measured by its ability to use the least quantity but
totally prevent Staphylococcus aureus in 50ml bouillon
culture-medium as 1Unit,After the purification of
penicillin,1Unit was definited to be 0.5988μg
benzylpenicillin sodium, so 0.5988μg benzylpenicillin
sodium was used as 1Unit penicillin,
Antibiotic mechanism of
action
（ 3） Drug resistance of microorganisms
Six mechanisms of drug resistance,
① change structure so that some drug cannot affect,In the case of penicillin,by
converting to a cell-wall-deficient form (L form) that penicillin cannot affect,
② decrease in cell permeability and uptake of the drug,Streptomyces venezuelae
prevents cyclomycin from entering cell by changing permeability of membrane,
③ Many bacteria possess multidrug resistant pumps that actively transport
drugs and other chemicals out of cells,Some resistant bacteria like E,coli and
Staphylococcus aureus can pump proton in and drug out of cell by proton/drug
exchanger in membrane,
④ synthesis of enzymes that inactivate the drug,For example,some
Staphylococcus aureus produce lactamase,which can break thelactam ring and lose
bacteriostatic action;
⑤ change in the number or affinity of the drug receptor sites,Because most
drugs act on a specific target such as protein,RNA,DNA,or membrane
structure,microbes can circumvent drugs by altering the nature of this target,
Phytomycin resistance is associated with an alteration on the 30S ribosomal binding
site,
⑥ modification of an essential metabolic pathway,Some strains resistant to
sulfonic acid alter the nature of dihydrofolate synthetase and synthesize an enzyme
which is insensitive to sulfonic acid,
Inherent resistance to antibiotic
Spread of resistance factors
References
Prescott LM,Harley JP,and Klein DA.,Microbiology (5th ed.),Higher education
press and McGraw-Hill Companies,Inc.2002
Michael TM,John MM,Jack P,Brock.,Biology of Micoorganisms,International
edition,Pearson Education,Inc.2003
Talaro K,P,Foundations in Microbiology (Fifth Edition),Higher education press
and McGraw-Hill Companies,Inc.2005
Zhou De-qing,Microbiology textbook,second edition,high education publishing
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