Chapter 6
The growth of microbe and its control
(微生物的生长及其控制 )
Growth：
Reproduction：
The relationship between individual and colony
colony growth=individual growth + individual reproduction
Section A
Determination of growth and reproduction
（测定生长繁殖的方法）
A,Determination of growth quantum（ quantity）,suitable
for all microbes.
(测定生长量 )
B,Counting reproduction number,only suitable for
unicelled bacteria and yeast,not for the filamentous
moulds and actinomyces.
(计繁殖数 )
A,Measuring of growth
quantity
（测生长量）
Direct measuring (直接法 )
1.volumetry
2,dry weight weighing
Indirect measuring(间接法 )
1,physiological index 生理指标法
nitrogen content
indexes carbon content
P,DNA,RNA,ATP,DAP,
chitin etal.
2,turbidimetry (nephelometry)比浊法
turbidimetry
B,Offspring counting
1.Direct ways(直接法 )
Using blood counting plate under the light microscope,
the result is the the total number including the dead cells,
living cells and dead cells will be distinguished if staining
with special dye before counting,thus both living cell
number and the total number can be obtained
respectively through this way,
Direct counting under light microscpe
Structure of the counting area
Colorless ( living)
Yeast (methylene blue)
Blue ( dead)
Bacteria stain with acridine orange (吖啶橙 ) and
observe under UV microscope
Orange fluorescence (living cell)
Green fluorescence (dead cell)
第三节 丝状微生物的生长图6 - 3 - 1 腐皮镰孢霉生长曲线
0
1
2
3
4
5
0 10 20 30 40
时间（h ）
干重（m
g
）
4
5
6
7
8
9
干重的自然对数（u
g
）
1,菌丝呈顶端生长
2,没有对数生长期
The growth of filamentous
microbe
2,Indirect ways (living cell counting)
The methods are designed on the basis that
living microbe can increase the turbidity while
growing in liquid medium and can form colony
on the solid medium,The most common used
method is colony-counting,
2.1 plate colony-counting methods(平板菌落计数法 )
The method is suitable for aerobic and anearobic
microbes,every living cell will form a colony forming unit
(菌落形成单位 cfu),the total number of living cell in the
sample can be calculated from cfu × dilution factor(稀释倍数 ).
pour plate (浇注平板 )
spread plate (涂布平板 )
225ml无菌水加 25g样品
1ml
1ml
1ml
试管中原始装
9ml无菌水
10-2 10-3 10-4
1ml
2.2 anaerobic colony-counting method
Hungate roll-tube technique (亨盖特滚管技术 ) is
designed by famous American microbiologist R,E,Hungate
in 1950,it’s a epoch-making invention in the development
history of microbiology which made the isolation and study
for the obligate anearobic possible.
The method above needs complicated equipment and the
skillful technique,Zhou deqing invented semi-solid media
deep agar method(半固体深层琼脂法 ) which is easily
manipulated.
Section B
Rules of microbial growth
Part A cell growth and synchronous growth
Part B typical growth curve of unicelled microbe
Part C continuous culture
Part D high cell-density culture
Part A
cell growth and synchronous growth
Extremely complicated biochemical changes and
cytological changes are going on in the tiny cells periodically
along with the cell growth,the observation of these changes
are fairly difficult,The methods used present are,
1,Ultra-thin section EM (电镜观察超薄切片 )
2,Synchronous culture (同步培养技术 )
Synchronous culture trying to keep a group of cells
synchronous in cell growth and at the same stages of cell
cycle,thus the individual biochemical changes can be
indirectly studied through the group.
Ways to keep synchronous growth:
1,Induced by the environmental condition(环境条件诱导法 )
2,Mechanically screening (机械筛选法 )
The sequence of events extending from the formation of a
new cell to the next division is called the cell cycle.(细胞周期是指从一个细胞分裂结束到第二次新的细胞分裂结束所经历的顺序事件 )
In this cycle,a growing E,coli will double in length then
divide into two cells of equal size,with each new cell
containing at least one copy of the bacterial DNA,
Consequently,during this time,a copy of the chromosome
must be synthesized and the two chromosomes segregated into
the two daughter cells,
Cell cycle
DNA replication occurs during the C (chromosome
replication) phase and chromosome segregation（分离）
occurs in the G (gap) phase,which may be of variable
length,Segregation of the chromosomes is achieved by
attachment of the replicated DNA onto two adjacent sites
on the membrane,Membrane growth between the sites
pushes the chromosomes towards the poles of the cells,
Finally,a cross wall (septum) is laid down between the
two chromosomes and the cell divides into two (D
phase).
DNA replication
Cell division and DNA replication have to be coordinated.
（协调） Initiation of DNA replication at the origin (oriC),a
short adenine and thymine rich sequence,is dependent on the
cell reaching a critical mass (initiation mass)（ DNA复制取决于细胞中起始物质所达到的临界量 （准备 DAN 复制相关的酶和蛋白） ）
and requires a number of protein initiation factors,DNA
segregation and division,however,are controlled by the
length of the cell which must reach a particular threshold
length（阈值） before the chromosomes are partitioned and
cell division initiated.
Coordimated replication
and division
Fig,Stages in the cell cycle of prokayotes.
Rapid growth
When conditions for growth are favorable,E,coli can
grow with a doubling time（生长倍增时间） of approximately
20 min,However,the time it takes to synthesize a complete
copy of the E,coli chromosome is 40 min,under optimum
conditions and segregation of the DNA and division takes
another 20 min,Thus,the shortest cell cycle and,therefore,
doubling time for E,coli should be 60 min,This is obviously
not the case,For cells to divide faster than every 60,min,
DNA replication must begin in one cycle and finish in
another,
Contradiction
When cells are growing fast (doubling time <60 min),
initiation of replication occurs,as normal,producing two
replication forks which move bidirectionally round the
chromosome to the termination point,However,the origins
on these new strands then initiate further rounds of replication
before the previous round of DNA replication has finished
(Fig.),Thus,when cell division occurs the DNA in the
daughter cells is already replicating,The faster the cell growth
rate,the more replication forks are formed such that the DNA
in new cells may have multiple replication forks,
Why?
That repeated DNA replication can occur without cell
division indicates that the control of the two processes
is not linked,This is in contrast to eukaryotic cells
where the two processes are directly connected,
Indications
Fig,2,Coordination of DNA synthesis and cell
division in slow and fast growing bacteria.
Slow growth Fast growth
origins
Replication
forks
Initiation of
DNA synthesis
DNA
synthesis
already
initiated in
new cells
Part B
Typical growth curve of unicelled
microbe
The best way of producing great numbers of microbes or their
natural products is to culture in the liquid medium,Normally,
the technique we use is called batch culture（分批培养） in
which the bacteria are inoculated into flasks(三角瓶 )of a
suitable medium and grown at an appropriate temperature and
degree of aeration（通气条件）,
Batch culture
Bacteria grown in this way show a particular pattern
of growth which is referred to as the bacterial growth
curve,The number of viable bacterial cells is measured
over time（整个时间） and is plotted as a graph of the
log10 viable cell numbers（活细胞数） against time,
which is called a semi-logarithmic plot（半对数关系曲线）,The bacterial growth curve reveals four phases
of growth.
Growth curve
A logarithmic scale（对数尺度） is used to plot bacterial
growth owing to the large numbers of cells produced and to
reveal the exponential nature of bacterial growth,If an
arithmetic scale（算术尺度） is used to plot the increase in
the number of cells,a curve of increasing gradient would be
seen which is converted into a straight line when a
logarithmic scale is used.（增加的剃度曲线变成直线）
The doubling time for the bacteria can be read directly from
the graph.
Why use a logarithmic scale
rather than arithmetic scale？
When bacteria are first inoculated into a medium there
is a period in which no growth occurs,During this phase
the bacteria are adapting to the new environment,
synthesizing new enzymes as required and increasing cell
size ready for cell division,The length of this time depends
on the nature of the inoculum(接种物,pl,inocula),If this
comes from a fresh culture in the same medium,the lag
phase will be short,but if the inoculum is old or the
medium has been changed (especially moving bacteria
from a rich medium to a poor one) the lag phase will be
longer.
1,Lag phase（延迟期）
Factors that affect the time of lag phase:
(1) The nature of the inoculum,fresh or old.(接种龄 )
(2) Seed volume or pitching rate.(接种量 )
(3) The ingredients of the medium.(培养基成分 )
Once the bacteria start to divide,the numbers
increase at a constant rate（恒速） which reflects the
doubling time of the bacteria,This is seen as a
straight line part of the graph.
2,Exponential (logarithmic) phase
（指数期或对数期）
This stage has the following features:
(1) Maximum R(生长速率常数 ),the shortest generation
time(代时 ) for cell fission or the shortest doubling
time(倍增时间 )for the doubling of the cytoplasm,
(2) Balanced growth of the cell,so the ingredient of the
cell is evenly distributed(细胞进行平衡生长,故菌体各部分成分均匀 )
(3) Active enzyme complex,blooming metabolism.(酶系活跃,代谢旺盛 )
Based on the advantages we discussed above,
the microbes of the whole group have relatively
consistent physiological attributes,therefore is the
better material for the metabolism and
physiological researches,and is the optimum host
cell to multiply the bacteriophage,the suitable
seed for the fermentation industry as well.
usages
Factors that affect the period of the
exponential phase:
(1) Bacterial species
(2) Ingredients of the medium
(3) Concentration of the nutrients
(4) Culture temperature
Escherichia coli 12.5~17min.
Bacillus subtulis 26~32
Lactobacillus acidphilus 66~87
Streptococcus lactis 26~48
The concentration of the nutrient can affect both the
growth rate (R) and the total amount of growth,The
growth rate only can be affected while the nutrients
concentration is rather low,When the nutrients
concentration is gradually increased,only the final cell
yield is affected and the growth rate isn’t affected,If the
concentration is further increased,both the growth rate
and the growth amount wouldn’t be affected any more,
Growth-limited factor
(生长限制因子 )
The growth-limited factor is a kind of
nutrient that can affect the growth rate and
the growth amount in a small range of
concentration.
(凡处于较低浓度范围内可影响生长速率和菌体产量的某营养物,就称生长限制因子 )
As the bacteria increase in numbers they use up all the
available nutrients and produce toxic waste products,
Eventually a point is reached where there is no net increase in
cell numbers,seen as a flattening off（变平的部分） of the
growth curve,During this state of equilibriumcells are still
functioning （在稳定阶段，细胞仍有功能）,There is
some cell death which is balanced by some small amounts of
controlled cell division.
3,Stationary phase
（稳定期或平衡期）
Once the cell growth enters the stationary phase,the inclusion
bodies(细胞内含物 )such as glucogen,metachromatic granules
and fat become to accumulate,and the spore become to form in
the bacillus,some microbes begin to synthesize the useful
secondary metabolites(次生代谢物 eg,antibiotics ) via complex
secondary metabolism pathway from the precursor—— primary
metabolites(初生代谢物 ),thus the secondary metabolites also is
called idiolites(稳定期产物 ).
Special product of the
stationary phase
Why the stationary phase
comes?
The reasons are:
(1) The exhaustion of the nutrients especially the growth-
limited factor.
(2) The ratio disorder of the nutrients particularly C/N ratio.
(3) The accumulation of the harmful metabolites for instance
the acid,alcohol toxin or H2O2
(4) the physiochemical conditions becomes more and more
unsuitable (pH,redox potential ).
The growth pattern of the stationary plays the
instructive role in the practice:
(1) it’s the optimum harvesting time for the fermentative
production designed for the bacteria or the bacteria-
growth paralleled metabolites.(产物的最佳收获期 )
(2) It’s the best bioassay time for the amino acid,
nitrogenous bases and amino acids.（ 最佳生物测定时期）
(3) The study on the reason about the coming of the
stationary phase accelerate the putting forward for the
principles of continuous culture and the development of
technology and techniques,(通过对稳定期到来原因的研究，
还促进了连续培养原理的提出和工艺、技术的创建 )
After a while the rate of cell death becomes greater than
cell division and the number of viable cells drops,The
whole group is in a negative-increasing state,The increasing
proteinase activity resulted in the autolysis of some
microbes and the culture becomes less turbid,some
beneficial secondary metabolites (antibiotics) are further
synthesized or released by some species in this stage,and
for the bacillus,spore is also released during this time.
4,Death phase（衰亡期）
A typical bacterial growth curve.
time
Part C microbial continuous culture
Continuous culture also named open culture,it
is put forward on the basis of batch culture or
closed culture in which the unicelled microbes
shows the typical growth curve,When
continuous culture is used in practice,it is called
continuous fermentation(连续发酵 ),
The problem with batch culture is that eventually cell
growth stops due to the exhaustion of nutrients or the
accumulation of toxic products,If these problems can be
prevented by replacing the spent medium with fresh,then
bacterial growth can be maintained indefinitely,
Continuous culture systems such as the chemostat do just
that,A major advantage of these systems is in the
provision of constant conditions for physiological studies,
This is impossible in batch culture as the growth of the
bacteria changes the environmental conditions such as pH
and nutrient concentration of the medium.
Batch culture versus
continuous culture
Batch culture Continuous culture
chemostat
turbidostat
Continuous
medium flow in
The relationship between batch culture and continuous culture
Time,late stage of
exponential phase
They are flow systems in which fresh medium is
constantly fed into a culture vessel which is kept at a
constant volume by an overflow(溢流 ) system that removes
the excess of liquid so the bacteria are kept in the
exponential phase of growth,Consequently,once the system
has reached equilibrium,cell numbers are kept constant and
a steady-state growth rate is reached,This growth rate can
be manipulated by altering conditions such as medium
composition and flow rate.
chemostat
c
flm
Culture
vessel
Overflow pipe to
collecting vessel
培养基流速控制搅拌器通入空气培养瓶 至收容器的流出管
Fig.4 a simplified diagram of a continuous culture system
Medium
storing vessel
filter Medium
inflow pipe
pump chemostat
Collecting vessel
filter
Sampling outlet
Stirring
mechanism
Air filter
Classification of Continuous culture apparatus
B one-step continuous fermenter
Steps
Multi-step continuous fermenter
A turbidostat(恒浊器 )
controlling style
chemostat(恒化器 )
单级连续培养器多级连续培养器
The comparison of turbidostat and chemostat
1.Contolling cell density flow rate of medium
object （ internal control） （ external control）
2.Medium without limit growth factor with
3,Medium flow rate nonconstant constant
4.Growth rate the highest slower than the highest
5.Product large number of bacteria cells of different
or cell paralleled metabolites growth rate
6.Appliance in the factory in the laboratory
one-step turbidostat continuous fermentor (单级恒浊式连续发酵器 )can be used for the production or research on the
condition that metabolite synthetic rate parallelled with the
bacterial growth rate,on the contrary,if the two rates is
inconsistant,for instance acetone,butyl alcohol(butanol丁醇 ),
and some secondary metabolites,multi-step continuous
fermenter should be designed and adopted,
One-step and multi-step continuous fermenter
eg,The fermentation of
acetone and butyl
alcohol
Trophophase (菌体生长期 37)
Clostridium acetobutylicum
idiophase（产物合成期 33）
The advantages of continuous fermentation
（ 1） high efficiency
（ 2） self-control
（ 3） steady quality
（ 4） economical
Part D
high cell-density culture
(HCDC,微生物的高密度培养 )
Sometimes called high cell-density fermentation,in this
technique the cell density in the liquid medium is over 10
times than that of normal culture,It is gradually developed
in the practice of producing multi-peptides medicine by
genetic engineering bacteria especially E.coli,These
medicine include human growth hormone,insulin,human
interferon,IL etal,
Significance
HCDC is economically significant in practice in that it
would increase the specific production rate （比生产率，
即单位体积单位时间内产物的产量）,it not only
decreased the fermentor volume,exhausting of the
medium ； increase the isolation and extraction rate in the
down-stream processing(下游工程 ),but also shortened
the production cycle,decrease the equipment input and
the production cost.
Ways of high cell-density culture
(1) Optimize the medium ingredients and its content
(选取最佳培养基成分和各成分含量 )
(2) Feeding (补料 Fed cultivation)
(3) Increase the concentration of dissolved oxygen
(提高溶解氧的浓度 )
(4) Preventing the formation of harmful metabolites
(防止有害代谢产物的生成 )
Section C
major factors involved in the
microbial growth
1,Tempereture
2,Oxygen
3,pH
Three cardinal point of growth
temperature (生长温度三基点 )
Minimum
optimum
maximum
Temperature
Psychrophiles such as Bacillus psychrophilus (嗜冷芽孢杆菌 ) are bacteria that have become adapted
to living at temperatures as low as -10° C and have
an optimum growth temperature around 20° C.
Psychrophiles(嗜冷微生物 )
Mesophiles(嗜温微生物 ) are bacteria that
grow at temperatures between 15° C and
45° C with an optimum around 37° C.
Mesophiles(嗜温微生物 )
Eubacterial thermophiles typified by B,
strearothermophilus (嗜热脂肪芽孢杆菌 )grow between 30° C and 75° C with
an optimum of 55° C.
thermophiles(嗜热微生物 )
There are also groups of archaebacteria that have
been isolated from hot-air vents in the seabed(海底火山喷气口 )which are capable of growth at
temperatures greater than 100° C,termed
hyperthermophiles(嗜高热微生物 ).
hyperthermophiles(嗜高热微生物 )
Bacteria vary in their requirements for oxygen,depending on
the nature of their metabolism,Aerobes are bacteria that are
capable of growing in the presence of oxygen; anaerobes are
bacteria that do not require oxygen for growth,Within this range
are obligate anaerobes that die in the presence O2; facultative
anaerobes such as E,coli which grow much better in the
presence of O2,but can grow anaerobically; aerotolerant
anaerobes that ignore the presence of O2 and grow equally well
in its presence or absence; and microaerophilic bacteria that are
damaged by normal atmospheric concentrations of 20% and
only survive at much lower concentrations of O2.
oxygen
Obligate or strict aerobes
Aerobes facultative anaerobes
Microaerophilic bacteria
Aerotolerent anaerobes
Anaerobes facultative aerobes
Obligate anaerobes
oxygen
5类对氧关系不同的微生物在半固体琼脂柱中的生长状态模式图
facultative
anaerobes
aerobes microaerophic
aerotolerant
anaerobes
Bacteria that can grow at high pH (8.5-11.5) are called
alkaliphiles,
Acidophiles are those that grow at low pH (0-5.5).
pH
microbes Minimum optimum maximum
pH spectrum of different microbes
Antibiotic-producing microbes growth antibiotic synthesis
The optimum pH for growth and antibiotic
synthesis
Possible reactions responsible for the pH
changing during the cultivation
Measures used in the fermentative
production to regulate the pH
Like all organisms microbes are sensitive to the
osmolarity of the surrounding medium,at low osmolarity
water will be accumulated in the cell and at high
osmolarity water will be lost,Lysis of the cell at low
osmolarity is prevented by the presence of the cell wall,
Growth at high osmolarity is dependent on the ability of
the microbe to maintain a high osmolarity within the cell
without dam aging cellular metabolism,
Water
activity
Compounds such as betaine,choline and potassium
ions (called compatible solutes) are used by
bacteria to maintain this osmotic balance,
Bacteria,such as Staphylococcus aureus,that are
capable of growing in 3 M NaCI,are called
osmotolerant whereas bacteria that have become
adapted to growth at very high concentrations of
salt (2.8-6.2 M NaCI),such as Halobacterium,are
called halophiles.
In the laboratory,water activity and pH are controlled by
the composition of the medium,Incubators are used to
provide the correct temperature,Aeration to provide
adequate 0; levels is normally achieved by shaking liquid
cultures or by providing large surface areas of solid
medium,Anaerobic conditions can be obtained by simply
filling a bottle to the top with medium so there is not much
room for air,but for more stringent anaerobes a reducing
agent such as thioglycolate can be added to the medium,
Specialist anaerobic jars or growth cabinets where 0^ can
be eliminated or replaced are also used.
Section D microbial culturing
(微生物培养法概论 )
Part A laboratory culturing
Part B microbial culturing apparatus in practice
Part A laboratory culturing
test-tube slant
Aerobes agar polate
Kolle flask
high agar column —— Veillon tube
brewer
anaerobic petri dishes spray
Anaerobes bray
hungate roll-tube technique
anaerobic jar
Anaerobic glove box
cover
Narrow space
Three kind of petri dishes for anaerobic
culture
section sketch
of anaerobic
test tube used
in Hungate
roll-tube
technique
剖面图
Section
figure of
anaerobic
jar
Anaerobic glove box
Mixed
gas
nitrogen
Connected to
vacuum pump
the appearance sketch of anaerobic glove box
Aerobes,the oxygen concentration is always the growth-
limited factor for aerobes,the following measures can be
taken to increase the concentration of the dissolved oxygen
in the medium:
(1) Superficial layer liquid stationary cultivation
(2) Shake-flask cultivation
(3) Ventilation in the bottom of deep liquid
(4) mechanically stirring
Aerobes cultivation in the laboratory,
（实验室常用液体培养法）
(1) Test tube cultivation （试管液体培养）
(2) Flask superficial layer cultivation（三角瓶浅层液体培养）
(3) Shake-flask cultivation （摇瓶培养）
(4) Benchtop fermentor (台式发酵罐 )
（ 1）（ 2） are only suitable for the facultative anaerobes
Liquid cultivation of anaerobes,
（ 1） anaerobic jar
（ 2） anaerobic glove box
（ 3） addition of reducing agent in the presence of oxygen
to decrease the redox potential.
(1),(2) needn’t any additional measures;
Reducing agent may be organic or inorganic
Microbial cultivation apparatus in practice
Solid cultivation
aerobes 曲法培养
anaerobes 堆积培养法
Liquid cultivation （ aerobes）
Shallow pan cultivation（浅盘培养）
Ventilated deep liquid cultivation（深层液体通气培养）
Diagram of ventilated qu（ moldy bran） slot
通风曲槽结构模式图
sky light
qu-making room
air vent
qu slot
Raw starter
篦架
fan 鼓风机
electromotor
通风曲
Widely used in the soy sauce brewing
The structure of a typical fermenter
Fermenter is the most
conventional bioreactor，
which is made of steel，
round barrel-shaped，
erective or stand-up
vessel with flat spherical
bottom and top（ lid)，
the ratio of the highness
to diameter is 1,2~2.5，
volume normally is
50~500 m3,the largest is
1500 m3 in Britan.
Ventilated deep liquid cultivation
发酵罐
Major functions of the fermenter
1,homologous nutrients
2.Well ventilation and stirring
3.Suitble temperature and pH
4,Eliminating the foam
5.Preventing the contamination of the unwanted microbes
典型发酵罐的构造
medium making
sterilization
medium in and feeding
product out extraction
inoculation
sampling analysis
waste gas out
bacteria free air in
heated water
(jacket)
cool water
Fermenter working
principles
Section E
The control of detrimental microbes
Part A several basic definitions
bacteriocidation
sterilization,thoroughly
Killing bacteriolysis
disinfection,partially
antisepsis,spoilage and mold
causing microbes
Inhibition
chemotherapy,pathogens in the host
Lo
g1
0 a
va
ila
ble
ce
lls
time
bacteriostasis bacteriocidation bacteriolysis
Total
living
Mesures are taken at the Logarithmic stage (arrow head )
Methods for antisepsis
Low temperature(0,-20,-70,-196℃ )
Shortage of oxygen
Dessication
High osmotic pressure
High acidity
High alcohol concentration
Antiseptic or preservative
Comparison of sterilization,disinfection,antisepsis and chemotherapy
items sterilizati
on
disinfection antisepsis chemothera
py
treatm
ent
target
microb
es
Effect
exampl
es
physiochem
ical (strong)
the whole
thing
all kinds of
thoroughly
killing
normal
autoclaving,
irradiation,
chemical
bcteriocide
Physiochemica
l (weak)
outside,liquor,
milk
associated
pathogen
killing or
inhibition
70% alcohol,
pasteurization
Physiochemica
l
Inside and
outside of
organics
all
Inhibition or
killing
Refrigeration,
cool storage,
salting,
oxygen
shortage,
chemical
preservatives
Chemotherapeut
ant
in host boby
associated
pathogen
inhibition or
killing
antibiotics,
Physical factors
1,High tempreture
2,Irradiation
3,Ultrasonic
4,Microwave
5,Laser
6,静高压
The most effective
The most convenient
Used the most often
Representation ——high temperature
Incineration（ or combustion）Dry heat sterilization
Heated air sterilization in oven
LTH 63℃,30min.
Pasterization
HTST 72℃,15 seconds
boiling 100℃,several minutes
tyndallization 80~100℃,15~60min,
37℃ overnight,3×
Normal autoclaving 121℃,15~20min.
High pressure
Continuous autoclaving 135~140℃,5~15mi.
atmospheric pressure
Moist heat
sterilization
Part B
quantification index used in heat sterilization
thermal death time,the shortest time to kill a certain
microbes suspended in the liquid at a certain temperature.
for example,E.coli,10min at 60℃,.
salmonella typhi 30min,at 58℃,
Thermal death point,the lowest temperature at which a
certain microbes suspended in the liquid are fully damaged in a
certain time (usually 10 min.)
For example,agrobacterium tumefaciens 53℃ for 10min.
Factors that effect normal autoclaving
1,amount of bacteria carried
2,air expulsion degree
3,pH of the object
4,volume of the object
5,heating and thermolysis rate
The effect of air eliminate
degree on the sterilization
temperature
pressure read by pressure gauge
LB/in2
Temperature in the autoclave
pure steam? air were eliminated without elimination
The detrimental effect of high temperature on the
medium ingredients
Organic eg,Polypeptides Formation of sediment
Inorganic eg,Phosphate and carbonate
browning (form amidoglycan,caramel,
melanin)
poisoning
pH changing
dropping of the Medium oncentration
nutrient damage
and color blacken
How to prevent
① use special heat sterilization
A,sterilizing respectively for saccharides and other ingredients,
phosphate and Ca2+,Fe3+
B,Sterilizing with low pressure
C,Fractional sterilization
D,continuous autoclaving
② filter sterilization
A,Filter membrane apparatus
B,Glass filter
C,Seitz filter
D,Chamberland filter
E,Berkefeld candle
③ others
Bacteria filter apparatus（细菌滤器）
Filtration
membrane
filter
Glass
filter
Seitz filter
（ asbestus）
Berkefeld
filter
Chamberland
filter
硅藻土过滤器 或贝氏过滤器磁土或张伯伦过滤器素烧瓷过滤器蔡氏滤器（石棉）烧结玻璃板过滤器
Part C
chemical bactericide,disinfectant and
chemotherapeutant
Liquid
surface disinfectant
gas
Chemical factors
antimetabolite (sulphonamides)
chemotherapeutant antibiotics
biopharmaceutin
Microbial antibiotic resistance
1,Formation of an enzyme inactivating the medicine
2,Modifying and changing the target site of the
medicine
3,Form the salvage pathway
4,Preventing the medicine across the cell membrane
5,Pump out the medicine by an active way
reasons
The growth of microbe and its control
(微生物的生长及其控制 )
Growth：
Reproduction：
The relationship between individual and colony
colony growth=individual growth + individual reproduction
Section A
Determination of growth and reproduction
（测定生长繁殖的方法）
A,Determination of growth quantum（ quantity）,suitable
for all microbes.
(测定生长量 )
B,Counting reproduction number,only suitable for
unicelled bacteria and yeast,not for the filamentous
moulds and actinomyces.
(计繁殖数 )
A,Measuring of growth
quantity
（测生长量）
Direct measuring (直接法 )
1.volumetry
2,dry weight weighing
Indirect measuring(间接法 )
1,physiological index 生理指标法
nitrogen content
indexes carbon content
P,DNA,RNA,ATP,DAP,
chitin etal.
2,turbidimetry (nephelometry)比浊法
turbidimetry
B,Offspring counting
1.Direct ways(直接法 )
Using blood counting plate under the light microscope,
the result is the the total number including the dead cells,
living cells and dead cells will be distinguished if staining
with special dye before counting,thus both living cell
number and the total number can be obtained
respectively through this way,
Direct counting under light microscpe
Structure of the counting area
Colorless ( living)
Yeast (methylene blue)
Blue ( dead)
Bacteria stain with acridine orange (吖啶橙 ) and
observe under UV microscope
Orange fluorescence (living cell)
Green fluorescence (dead cell)
第三节 丝状微生物的生长图6 - 3 - 1 腐皮镰孢霉生长曲线
0
1
2
3
4
5
0 10 20 30 40
时间（h ）
干重（m
g
）
4
5
6
7
8
9
干重的自然对数（u
g
）
1,菌丝呈顶端生长
2,没有对数生长期
The growth of filamentous
microbe
2,Indirect ways (living cell counting)
The methods are designed on the basis that
living microbe can increase the turbidity while
growing in liquid medium and can form colony
on the solid medium,The most common used
method is colony-counting,
2.1 plate colony-counting methods(平板菌落计数法 )
The method is suitable for aerobic and anearobic
microbes,every living cell will form a colony forming unit
(菌落形成单位 cfu),the total number of living cell in the
sample can be calculated from cfu × dilution factor(稀释倍数 ).
pour plate (浇注平板 )
spread plate (涂布平板 )
225ml无菌水加 25g样品
1ml
1ml
1ml
试管中原始装
9ml无菌水
10-2 10-3 10-4
1ml
2.2 anaerobic colony-counting method
Hungate roll-tube technique (亨盖特滚管技术 ) is
designed by famous American microbiologist R,E,Hungate
in 1950,it’s a epoch-making invention in the development
history of microbiology which made the isolation and study
for the obligate anearobic possible.
The method above needs complicated equipment and the
skillful technique,Zhou deqing invented semi-solid media
deep agar method(半固体深层琼脂法 ) which is easily
manipulated.
Section B
Rules of microbial growth
Part A cell growth and synchronous growth
Part B typical growth curve of unicelled microbe
Part C continuous culture
Part D high cell-density culture
Part A
cell growth and synchronous growth
Extremely complicated biochemical changes and
cytological changes are going on in the tiny cells periodically
along with the cell growth,the observation of these changes
are fairly difficult,The methods used present are,
1,Ultra-thin section EM (电镜观察超薄切片 )
2,Synchronous culture (同步培养技术 )
Synchronous culture trying to keep a group of cells
synchronous in cell growth and at the same stages of cell
cycle,thus the individual biochemical changes can be
indirectly studied through the group.
Ways to keep synchronous growth:
1,Induced by the environmental condition(环境条件诱导法 )
2,Mechanically screening (机械筛选法 )
The sequence of events extending from the formation of a
new cell to the next division is called the cell cycle.(细胞周期是指从一个细胞分裂结束到第二次新的细胞分裂结束所经历的顺序事件 )
In this cycle,a growing E,coli will double in length then
divide into two cells of equal size,with each new cell
containing at least one copy of the bacterial DNA,
Consequently,during this time,a copy of the chromosome
must be synthesized and the two chromosomes segregated into
the two daughter cells,
Cell cycle
DNA replication occurs during the C (chromosome
replication) phase and chromosome segregation（分离）
occurs in the G (gap) phase,which may be of variable
length,Segregation of the chromosomes is achieved by
attachment of the replicated DNA onto two adjacent sites
on the membrane,Membrane growth between the sites
pushes the chromosomes towards the poles of the cells,
Finally,a cross wall (septum) is laid down between the
two chromosomes and the cell divides into two (D
phase).
DNA replication
Cell division and DNA replication have to be coordinated.
（协调） Initiation of DNA replication at the origin (oriC),a
short adenine and thymine rich sequence,is dependent on the
cell reaching a critical mass (initiation mass)（ DNA复制取决于细胞中起始物质所达到的临界量 （准备 DAN 复制相关的酶和蛋白） ）
and requires a number of protein initiation factors,DNA
segregation and division,however,are controlled by the
length of the cell which must reach a particular threshold
length（阈值） before the chromosomes are partitioned and
cell division initiated.
Coordimated replication
and division
Fig,Stages in the cell cycle of prokayotes.
Rapid growth
When conditions for growth are favorable,E,coli can
grow with a doubling time（生长倍增时间） of approximately
20 min,However,the time it takes to synthesize a complete
copy of the E,coli chromosome is 40 min,under optimum
conditions and segregation of the DNA and division takes
another 20 min,Thus,the shortest cell cycle and,therefore,
doubling time for E,coli should be 60 min,This is obviously
not the case,For cells to divide faster than every 60,min,
DNA replication must begin in one cycle and finish in
another,
Contradiction
When cells are growing fast (doubling time <60 min),
initiation of replication occurs,as normal,producing two
replication forks which move bidirectionally round the
chromosome to the termination point,However,the origins
on these new strands then initiate further rounds of replication
before the previous round of DNA replication has finished
(Fig.),Thus,when cell division occurs the DNA in the
daughter cells is already replicating,The faster the cell growth
rate,the more replication forks are formed such that the DNA
in new cells may have multiple replication forks,
Why?
That repeated DNA replication can occur without cell
division indicates that the control of the two processes
is not linked,This is in contrast to eukaryotic cells
where the two processes are directly connected,
Indications
Fig,2,Coordination of DNA synthesis and cell
division in slow and fast growing bacteria.
Slow growth Fast growth
origins
Replication
forks
Initiation of
DNA synthesis
DNA
synthesis
already
initiated in
new cells
Part B
Typical growth curve of unicelled
microbe
The best way of producing great numbers of microbes or their
natural products is to culture in the liquid medium,Normally,
the technique we use is called batch culture（分批培养） in
which the bacteria are inoculated into flasks(三角瓶 )of a
suitable medium and grown at an appropriate temperature and
degree of aeration（通气条件）,
Batch culture
Bacteria grown in this way show a particular pattern
of growth which is referred to as the bacterial growth
curve,The number of viable bacterial cells is measured
over time（整个时间） and is plotted as a graph of the
log10 viable cell numbers（活细胞数） against time,
which is called a semi-logarithmic plot（半对数关系曲线）,The bacterial growth curve reveals four phases
of growth.
Growth curve
A logarithmic scale（对数尺度） is used to plot bacterial
growth owing to the large numbers of cells produced and to
reveal the exponential nature of bacterial growth,If an
arithmetic scale（算术尺度） is used to plot the increase in
the number of cells,a curve of increasing gradient would be
seen which is converted into a straight line when a
logarithmic scale is used.（增加的剃度曲线变成直线）
The doubling time for the bacteria can be read directly from
the graph.
Why use a logarithmic scale
rather than arithmetic scale？
When bacteria are first inoculated into a medium there
is a period in which no growth occurs,During this phase
the bacteria are adapting to the new environment,
synthesizing new enzymes as required and increasing cell
size ready for cell division,The length of this time depends
on the nature of the inoculum(接种物,pl,inocula),If this
comes from a fresh culture in the same medium,the lag
phase will be short,but if the inoculum is old or the
medium has been changed (especially moving bacteria
from a rich medium to a poor one) the lag phase will be
longer.
1,Lag phase（延迟期）
Factors that affect the time of lag phase:
(1) The nature of the inoculum,fresh or old.(接种龄 )
(2) Seed volume or pitching rate.(接种量 )
(3) The ingredients of the medium.(培养基成分 )
Once the bacteria start to divide,the numbers
increase at a constant rate（恒速） which reflects the
doubling time of the bacteria,This is seen as a
straight line part of the graph.
2,Exponential (logarithmic) phase
（指数期或对数期）
This stage has the following features:
(1) Maximum R(生长速率常数 ),the shortest generation
time(代时 ) for cell fission or the shortest doubling
time(倍增时间 )for the doubling of the cytoplasm,
(2) Balanced growth of the cell,so the ingredient of the
cell is evenly distributed(细胞进行平衡生长,故菌体各部分成分均匀 )
(3) Active enzyme complex,blooming metabolism.(酶系活跃,代谢旺盛 )
Based on the advantages we discussed above,
the microbes of the whole group have relatively
consistent physiological attributes,therefore is the
better material for the metabolism and
physiological researches,and is the optimum host
cell to multiply the bacteriophage,the suitable
seed for the fermentation industry as well.
usages
Factors that affect the period of the
exponential phase:
(1) Bacterial species
(2) Ingredients of the medium
(3) Concentration of the nutrients
(4) Culture temperature
Escherichia coli 12.5~17min.
Bacillus subtulis 26~32
Lactobacillus acidphilus 66~87
Streptococcus lactis 26~48
The concentration of the nutrient can affect both the
growth rate (R) and the total amount of growth,The
growth rate only can be affected while the nutrients
concentration is rather low,When the nutrients
concentration is gradually increased,only the final cell
yield is affected and the growth rate isn’t affected,If the
concentration is further increased,both the growth rate
and the growth amount wouldn’t be affected any more,
Growth-limited factor
(生长限制因子 )
The growth-limited factor is a kind of
nutrient that can affect the growth rate and
the growth amount in a small range of
concentration.
(凡处于较低浓度范围内可影响生长速率和菌体产量的某营养物,就称生长限制因子 )
As the bacteria increase in numbers they use up all the
available nutrients and produce toxic waste products,
Eventually a point is reached where there is no net increase in
cell numbers,seen as a flattening off（变平的部分） of the
growth curve,During this state of equilibriumcells are still
functioning （在稳定阶段，细胞仍有功能）,There is
some cell death which is balanced by some small amounts of
controlled cell division.
3,Stationary phase
（稳定期或平衡期）
Once the cell growth enters the stationary phase,the inclusion
bodies(细胞内含物 )such as glucogen,metachromatic granules
and fat become to accumulate,and the spore become to form in
the bacillus,some microbes begin to synthesize the useful
secondary metabolites(次生代谢物 eg,antibiotics ) via complex
secondary metabolism pathway from the precursor—— primary
metabolites(初生代谢物 ),thus the secondary metabolites also is
called idiolites(稳定期产物 ).
Special product of the
stationary phase
Why the stationary phase
comes?
The reasons are:
(1) The exhaustion of the nutrients especially the growth-
limited factor.
(2) The ratio disorder of the nutrients particularly C/N ratio.
(3) The accumulation of the harmful metabolites for instance
the acid,alcohol toxin or H2O2
(4) the physiochemical conditions becomes more and more
unsuitable (pH,redox potential ).
The growth pattern of the stationary plays the
instructive role in the practice:
(1) it’s the optimum harvesting time for the fermentative
production designed for the bacteria or the bacteria-
growth paralleled metabolites.(产物的最佳收获期 )
(2) It’s the best bioassay time for the amino acid,
nitrogenous bases and amino acids.（ 最佳生物测定时期）
(3) The study on the reason about the coming of the
stationary phase accelerate the putting forward for the
principles of continuous culture and the development of
technology and techniques,(通过对稳定期到来原因的研究，
还促进了连续培养原理的提出和工艺、技术的创建 )
After a while the rate of cell death becomes greater than
cell division and the number of viable cells drops,The
whole group is in a negative-increasing state,The increasing
proteinase activity resulted in the autolysis of some
microbes and the culture becomes less turbid,some
beneficial secondary metabolites (antibiotics) are further
synthesized or released by some species in this stage,and
for the bacillus,spore is also released during this time.
4,Death phase（衰亡期）
A typical bacterial growth curve.
time
Part C microbial continuous culture
Continuous culture also named open culture,it
is put forward on the basis of batch culture or
closed culture in which the unicelled microbes
shows the typical growth curve,When
continuous culture is used in practice,it is called
continuous fermentation(连续发酵 ),
The problem with batch culture is that eventually cell
growth stops due to the exhaustion of nutrients or the
accumulation of toxic products,If these problems can be
prevented by replacing the spent medium with fresh,then
bacterial growth can be maintained indefinitely,
Continuous culture systems such as the chemostat do just
that,A major advantage of these systems is in the
provision of constant conditions for physiological studies,
This is impossible in batch culture as the growth of the
bacteria changes the environmental conditions such as pH
and nutrient concentration of the medium.
Batch culture versus
continuous culture
Batch culture Continuous culture
chemostat
turbidostat
Continuous
medium flow in
The relationship between batch culture and continuous culture
Time,late stage of
exponential phase
They are flow systems in which fresh medium is
constantly fed into a culture vessel which is kept at a
constant volume by an overflow(溢流 ) system that removes
the excess of liquid so the bacteria are kept in the
exponential phase of growth,Consequently,once the system
has reached equilibrium,cell numbers are kept constant and
a steady-state growth rate is reached,This growth rate can
be manipulated by altering conditions such as medium
composition and flow rate.
chemostat
c
flm
Culture
vessel
Overflow pipe to
collecting vessel
培养基流速控制搅拌器通入空气培养瓶 至收容器的流出管
Fig.4 a simplified diagram of a continuous culture system
Medium
storing vessel
filter Medium
inflow pipe
pump chemostat
Collecting vessel
filter
Sampling outlet
Stirring
mechanism
Air filter
Classification of Continuous culture apparatus
B one-step continuous fermenter
Steps
Multi-step continuous fermenter
A turbidostat(恒浊器 )
controlling style
chemostat(恒化器 )
单级连续培养器多级连续培养器
The comparison of turbidostat and chemostat
1.Contolling cell density flow rate of medium
object （ internal control） （ external control）
2.Medium without limit growth factor with
3,Medium flow rate nonconstant constant
4.Growth rate the highest slower than the highest
5.Product large number of bacteria cells of different
or cell paralleled metabolites growth rate
6.Appliance in the factory in the laboratory
one-step turbidostat continuous fermentor (单级恒浊式连续发酵器 )can be used for the production or research on the
condition that metabolite synthetic rate parallelled with the
bacterial growth rate,on the contrary,if the two rates is
inconsistant,for instance acetone,butyl alcohol(butanol丁醇 ),
and some secondary metabolites,multi-step continuous
fermenter should be designed and adopted,
One-step and multi-step continuous fermenter
eg,The fermentation of
acetone and butyl
alcohol
Trophophase (菌体生长期 37)
Clostridium acetobutylicum
idiophase（产物合成期 33）
The advantages of continuous fermentation
（ 1） high efficiency
（ 2） self-control
（ 3） steady quality
（ 4） economical
Part D
high cell-density culture
(HCDC,微生物的高密度培养 )
Sometimes called high cell-density fermentation,in this
technique the cell density in the liquid medium is over 10
times than that of normal culture,It is gradually developed
in the practice of producing multi-peptides medicine by
genetic engineering bacteria especially E.coli,These
medicine include human growth hormone,insulin,human
interferon,IL etal,
Significance
HCDC is economically significant in practice in that it
would increase the specific production rate （比生产率，
即单位体积单位时间内产物的产量）,it not only
decreased the fermentor volume,exhausting of the
medium ； increase the isolation and extraction rate in the
down-stream processing(下游工程 ),but also shortened
the production cycle,decrease the equipment input and
the production cost.
Ways of high cell-density culture
(1) Optimize the medium ingredients and its content
(选取最佳培养基成分和各成分含量 )
(2) Feeding (补料 Fed cultivation)
(3) Increase the concentration of dissolved oxygen
(提高溶解氧的浓度 )
(4) Preventing the formation of harmful metabolites
(防止有害代谢产物的生成 )
Section C
major factors involved in the
microbial growth
1,Tempereture
2,Oxygen
3,pH
Three cardinal point of growth
temperature (生长温度三基点 )
Minimum
optimum
maximum
Temperature
Psychrophiles such as Bacillus psychrophilus (嗜冷芽孢杆菌 ) are bacteria that have become adapted
to living at temperatures as low as -10° C and have
an optimum growth temperature around 20° C.
Psychrophiles(嗜冷微生物 )
Mesophiles(嗜温微生物 ) are bacteria that
grow at temperatures between 15° C and
45° C with an optimum around 37° C.
Mesophiles(嗜温微生物 )
Eubacterial thermophiles typified by B,
strearothermophilus (嗜热脂肪芽孢杆菌 )grow between 30° C and 75° C with
an optimum of 55° C.
thermophiles(嗜热微生物 )
There are also groups of archaebacteria that have
been isolated from hot-air vents in the seabed(海底火山喷气口 )which are capable of growth at
temperatures greater than 100° C,termed
hyperthermophiles(嗜高热微生物 ).
hyperthermophiles(嗜高热微生物 )
Bacteria vary in their requirements for oxygen,depending on
the nature of their metabolism,Aerobes are bacteria that are
capable of growing in the presence of oxygen; anaerobes are
bacteria that do not require oxygen for growth,Within this range
are obligate anaerobes that die in the presence O2; facultative
anaerobes such as E,coli which grow much better in the
presence of O2,but can grow anaerobically; aerotolerant
anaerobes that ignore the presence of O2 and grow equally well
in its presence or absence; and microaerophilic bacteria that are
damaged by normal atmospheric concentrations of 20% and
only survive at much lower concentrations of O2.
oxygen
Obligate or strict aerobes
Aerobes facultative anaerobes
Microaerophilic bacteria
Aerotolerent anaerobes
Anaerobes facultative aerobes
Obligate anaerobes
oxygen
5类对氧关系不同的微生物在半固体琼脂柱中的生长状态模式图
facultative
anaerobes
aerobes microaerophic
aerotolerant
anaerobes
Bacteria that can grow at high pH (8.5-11.5) are called
alkaliphiles,
Acidophiles are those that grow at low pH (0-5.5).
pH
microbes Minimum optimum maximum
pH spectrum of different microbes
Antibiotic-producing microbes growth antibiotic synthesis
The optimum pH for growth and antibiotic
synthesis
Possible reactions responsible for the pH
changing during the cultivation
Measures used in the fermentative
production to regulate the pH
Like all organisms microbes are sensitive to the
osmolarity of the surrounding medium,at low osmolarity
water will be accumulated in the cell and at high
osmolarity water will be lost,Lysis of the cell at low
osmolarity is prevented by the presence of the cell wall,
Growth at high osmolarity is dependent on the ability of
the microbe to maintain a high osmolarity within the cell
without dam aging cellular metabolism,
Water
activity
Compounds such as betaine,choline and potassium
ions (called compatible solutes) are used by
bacteria to maintain this osmotic balance,
Bacteria,such as Staphylococcus aureus,that are
capable of growing in 3 M NaCI,are called
osmotolerant whereas bacteria that have become
adapted to growth at very high concentrations of
salt (2.8-6.2 M NaCI),such as Halobacterium,are
called halophiles.
In the laboratory,water activity and pH are controlled by
the composition of the medium,Incubators are used to
provide the correct temperature,Aeration to provide
adequate 0; levels is normally achieved by shaking liquid
cultures or by providing large surface areas of solid
medium,Anaerobic conditions can be obtained by simply
filling a bottle to the top with medium so there is not much
room for air,but for more stringent anaerobes a reducing
agent such as thioglycolate can be added to the medium,
Specialist anaerobic jars or growth cabinets where 0^ can
be eliminated or replaced are also used.
Section D microbial culturing
(微生物培养法概论 )
Part A laboratory culturing
Part B microbial culturing apparatus in practice
Part A laboratory culturing
test-tube slant
Aerobes agar polate
Kolle flask
high agar column —— Veillon tube
brewer
anaerobic petri dishes spray
Anaerobes bray
hungate roll-tube technique
anaerobic jar
Anaerobic glove box
cover
Narrow space
Three kind of petri dishes for anaerobic
culture
section sketch
of anaerobic
test tube used
in Hungate
roll-tube
technique
剖面图
Section
figure of
anaerobic
jar
Anaerobic glove box
Mixed
gas
nitrogen
Connected to
vacuum pump
the appearance sketch of anaerobic glove box
Aerobes,the oxygen concentration is always the growth-
limited factor for aerobes,the following measures can be
taken to increase the concentration of the dissolved oxygen
in the medium:
(1) Superficial layer liquid stationary cultivation
(2) Shake-flask cultivation
(3) Ventilation in the bottom of deep liquid
(4) mechanically stirring
Aerobes cultivation in the laboratory,
（实验室常用液体培养法）
(1) Test tube cultivation （试管液体培养）
(2) Flask superficial layer cultivation（三角瓶浅层液体培养）
(3) Shake-flask cultivation （摇瓶培养）
(4) Benchtop fermentor (台式发酵罐 )
（ 1）（ 2） are only suitable for the facultative anaerobes
Liquid cultivation of anaerobes,
（ 1） anaerobic jar
（ 2） anaerobic glove box
（ 3） addition of reducing agent in the presence of oxygen
to decrease the redox potential.
(1),(2) needn’t any additional measures;
Reducing agent may be organic or inorganic
Microbial cultivation apparatus in practice
Solid cultivation
aerobes 曲法培养
anaerobes 堆积培养法
Liquid cultivation （ aerobes）
Shallow pan cultivation（浅盘培养）
Ventilated deep liquid cultivation（深层液体通气培养）
Diagram of ventilated qu（ moldy bran） slot
通风曲槽结构模式图
sky light
qu-making room
air vent
qu slot
Raw starter
篦架
fan 鼓风机
electromotor
通风曲
Widely used in the soy sauce brewing
The structure of a typical fermenter
Fermenter is the most
conventional bioreactor，
which is made of steel，
round barrel-shaped，
erective or stand-up
vessel with flat spherical
bottom and top（ lid)，
the ratio of the highness
to diameter is 1,2~2.5，
volume normally is
50~500 m3,the largest is
1500 m3 in Britan.
Ventilated deep liquid cultivation
发酵罐
Major functions of the fermenter
1,homologous nutrients
2.Well ventilation and stirring
3.Suitble temperature and pH
4,Eliminating the foam
5.Preventing the contamination of the unwanted microbes
典型发酵罐的构造
medium making
sterilization
medium in and feeding
product out extraction
inoculation
sampling analysis
waste gas out
bacteria free air in
heated water
(jacket)
cool water
Fermenter working
principles
Section E
The control of detrimental microbes
Part A several basic definitions
bacteriocidation
sterilization,thoroughly
Killing bacteriolysis
disinfection,partially
antisepsis,spoilage and mold
causing microbes
Inhibition
chemotherapy,pathogens in the host
Lo
g1
0 a
va
ila
ble
ce
lls
time
bacteriostasis bacteriocidation bacteriolysis
Total
living
Mesures are taken at the Logarithmic stage (arrow head )
Methods for antisepsis
Low temperature(0,-20,-70,-196℃ )
Shortage of oxygen
Dessication
High osmotic pressure
High acidity
High alcohol concentration
Antiseptic or preservative
Comparison of sterilization,disinfection,antisepsis and chemotherapy
items sterilizati
on
disinfection antisepsis chemothera
py
treatm
ent
target
microb
es
Effect
exampl
es
physiochem
ical (strong)
the whole
thing
all kinds of
thoroughly
killing
normal
autoclaving,
irradiation,
chemical
bcteriocide
Physiochemica
l (weak)
outside,liquor,
milk
associated
pathogen
killing or
inhibition
70% alcohol,
pasteurization
Physiochemica
l
Inside and
outside of
organics
all
Inhibition or
killing
Refrigeration,
cool storage,
salting,
oxygen
shortage,
chemical
preservatives
Chemotherapeut
ant
in host boby
associated
pathogen
inhibition or
killing
antibiotics,
Physical factors
1,High tempreture
2,Irradiation
3,Ultrasonic
4,Microwave
5,Laser
6,静高压
The most effective
The most convenient
Used the most often
Representation ——high temperature
Incineration（ or combustion）Dry heat sterilization
Heated air sterilization in oven
LTH 63℃,30min.
Pasterization
HTST 72℃,15 seconds
boiling 100℃,several minutes
tyndallization 80~100℃,15~60min,
37℃ overnight,3×
Normal autoclaving 121℃,15~20min.
High pressure
Continuous autoclaving 135~140℃,5~15mi.
atmospheric pressure
Moist heat
sterilization
Part B
quantification index used in heat sterilization
thermal death time,the shortest time to kill a certain
microbes suspended in the liquid at a certain temperature.
for example,E.coli,10min at 60℃,.
salmonella typhi 30min,at 58℃,
Thermal death point,the lowest temperature at which a
certain microbes suspended in the liquid are fully damaged in a
certain time (usually 10 min.)
For example,agrobacterium tumefaciens 53℃ for 10min.
Factors that effect normal autoclaving
1,amount of bacteria carried
2,air expulsion degree
3,pH of the object
4,volume of the object
5,heating and thermolysis rate
The effect of air eliminate
degree on the sterilization
temperature
pressure read by pressure gauge
LB/in2
Temperature in the autoclave
pure steam? air were eliminated without elimination
The detrimental effect of high temperature on the
medium ingredients
Organic eg,Polypeptides Formation of sediment
Inorganic eg,Phosphate and carbonate
browning (form amidoglycan,caramel,
melanin)
poisoning
pH changing
dropping of the Medium oncentration
nutrient damage
and color blacken
How to prevent
① use special heat sterilization
A,sterilizing respectively for saccharides and other ingredients,
phosphate and Ca2+,Fe3+
B,Sterilizing with low pressure
C,Fractional sterilization
D,continuous autoclaving
② filter sterilization
A,Filter membrane apparatus
B,Glass filter
C,Seitz filter
D,Chamberland filter
E,Berkefeld candle
③ others
Bacteria filter apparatus（细菌滤器）
Filtration
membrane
filter
Glass
filter
Seitz filter
（ asbestus）
Berkefeld
filter
Chamberland
filter
硅藻土过滤器 或贝氏过滤器磁土或张伯伦过滤器素烧瓷过滤器蔡氏滤器（石棉）烧结玻璃板过滤器
Part C
chemical bactericide,disinfectant and
chemotherapeutant
Liquid
surface disinfectant
gas
Chemical factors
antimetabolite (sulphonamides)
chemotherapeutant antibiotics
biopharmaceutin
Microbial antibiotic resistance
1,Formation of an enzyme inactivating the medicine
2,Modifying and changing the target site of the
medicine
3,Form the salvage pathway
4,Preventing the medicine across the cell membrane
5,Pump out the medicine by an active way
reasons
