The impact of microorganisms on human affairs
Important Persons in Microbiology History
1684 Antonie van Leeuwenhoek (discovery of bacteria)
1857-1864 Louis Pasteur (lactic acid fermentation,yeast
alcohol fermentation,spontaneous generation theory
“swan neck flasks”)
1881-1884 Robert Koch (pure culture,cause of
tuberculosis,Koch’s postulates,cause of (霍乱 )
cholera）
1889 Martinus Beijerinck (concept of a virus)
1929 Alexander Fleming (discovery of penicilin)
1953 James Watson,Francis Crick (DNA structure)
1983 Luc Montagnier (discovery of HIV)
Prokaryotic Cell (versus
Eukaryotic Cell)
? Not compartmentalized
? Cell membranes lack sterols (e.g,cholesterol)
? Single circular chromosome
? Ribosomal are 70S with
– subunits 30S (16S rRNA)
– 50S (5S & 23S rRNA)
Coccus or Cocci
Rod
Spirillum or Spirilla
Spirochete
Budding and appendaged
Filamentous
2.Shape and arrangement
Procaryoic cell organization
Mesosome,
invaginationa of the plasma membrane
in the shape of vesicles,tubules,or lamellae.
Functions:
1.cell wall formation during division.
2.play a role in chromosome replicaton
and distribution to daughter cells.
Making Wall-less forms
? Result from action of:
–enzymes lytic for cell wall
–antibiotics inhibiting peptidoglycan
biosynthesis
? Wall-less bacteria that don’t replicate:
–spheroplasts (with outer membrane)?
–protoplasts (no outer membrane),?
? Wall-less bacteria that replicate
–L forms
Naturally Wall-less Genus
Mycloplasma
Within the cytoplasm of procaryotic (and eucaryotic)
cells are several kinds of reserve deposits,known as
inclusions,Some inclusions are common to a wide
variety of bacteria,whereas others are limited to a
small number of species and therefore serve as a
basis for identification,Among the more prominent
bacterial inclusions are the following:
Carbon storage polymers – PHB and glycogen
Phosphate polymers
Sulfur Granules
Gas Vacuoles
INCLUSIONS
Peptidoglycan
Muramic acid
Glucosamine
L-alanine
D-glutamic acid
L-lysine/Diaminopimelic acid
D-alanine
D-alanine
The mechanism of Gram staining
During the procedure the bacteria are first stained with
crystal violet and next treated with iodine to promote dye
retention,When gram-positive bacteria then are decolorized
with ethanol,the alcohol is thought to shrink the pores of the
thick peptidoglycan,Thus the dye-iodine complex is retained
during the short decolorization step and the bacteria remain
purple,In contrast,gram-negative bacteria peptidoglycan is
very thin,not as highly cross-linked,and has larger pores,
Alcohol treatment also may extract enough lipid from the
gram-neaative wall to increase its porosity further,For these
reasons,alcohol more readily removes the purple crystal
violet-iodine complex from gram-negative bacteria.
Special structure
? Pili and fimbriae
? Flagella
? The bacterial endospore
Chapter 1
The structure and function of prokaryotes
Pili and fimbriae
G-,short,fine,hairlike appendages
that are not involved in motility.
Fimbriae/fimbria:1000piece/cell,
adhesion to host epithelium
Sex pili/pilus,1-10piece/cell,
sexual conjugation,they are
genetically determined by sex factors
or conjugative plasmids and are required
for bacterial mating,
Receptors of some bacterial viruses.
Flagellar Arrangements
Amphitrichous
Peritrichous
Polar/monotrichous
Lophotrichous
Flagellar ultrastructure
Filament,from cell surface to the tip
Basal body,embedded in the cell
Hook,link the filament to its basal body
Basal body,
G+,
S (peptidoglycan),
M(plasma membrane)
G-,
L (out membrane),
P (peptidoglycan layer),
S-M(contact the plasma membrane)
The E,coli
Flagellar
Structure
Bacterial Behavior
Chemotaxis,Phototaxis,and Other Taxes
? Prokaryotes encounter gradients of physical
and chemical agents in nature,and the motility
machinery in the cell is designed to respond in
a positive or negative way to these gradients
by directing the movement of the cell either
toward or away from the signal molecule,
respectively.
? Such directed movements are called TAXES.
? Chemotaxis,a response to chemicals.
? Phototaxis,a response to light.
How to judge wether a cell
have flagella
? 1,Light Microscope by staining
? 2,Electron microscope
? 3,Feature of colony
? 4,Culture in semi-solid medium
Endospore Structure
Mature free endospore
Spore formation,lack of nutrients,7 stages
The mechnism of heat and other
kethal agents resistantance
1,Calcium dipicolinic acid
2,Heat stable enzyme
3,High G+C%
4,Less water
5,Amino acid containing surfur(cys)
Paraspore crystal as biopesticide
Importance of fungi
? 1,decomposers
? 2,Major cause of plant diseases
? 3,Many diseases of animals and humans are caused by
fungi
? 4,products,the making of bread,wine,beer(yeasts);
the preparation of some cheeses,soy sause and sufu; the
commercial production
of many organic acids and certain drugs; The
manufacture of many antibiotics and
immunosuppressive drug cyclosporine.
? 5,Important research tools in the study of fundamental
biological processes.
YM shift
? Many fungi that cause diseases in humans and
animals,are dimorphic,they have two forms.
? 1,The yeast (Y) form in the animals
? 2,The mold or mycelial form (M) in the external
enviroment.
? Plant-associated fungi,the mycelial form in the
plantl the yeast form in the external enviroment.
yeasts
? Unicellular fungus,spherical to egg
shaped,single nucleus and
repriduces either asexually by
budding and transverse division or
sexually throough spore formation
? No flagella,possess most of the other
eucaryotic organelles..
Cilia and flagella structure
? Fig4.24p90
Chapter3
Microbial nutrition
1.Nutrient requirement
2.Nutritional types of microorganisms
3.Uptake of nutrients
4.Culture media
Growth Factors
Amino acids are needed for protein synthesis,
purines and pyrimidines for nucleic acid synthesis,
Vitamins are small organic molecules that usually
make up all or part enzyme cofactors,and only
very small amounts are required for growth,
(1)amino acids,(2) purines and pyrimidines,(3) vitamins
Major nutritional
type
Sources of energy,
hydrogen/electrons,
and carbon
Representative
microorganisms
Photoautotroph
(Photolithotroph)
Light energy,inorganic
hydrogen/electron(H/e-)
donor,CO2 carbon source
Algae,Purple and green
bacteria,Cyanobacteria
Photoheterotroph
(Photoorganotroph)
Light energy,inorganic
H/e- donor,
Organic carbon source
Purple nonsulfur bacteria,
Green sulfur bacteria
Chemoautotroph
(Chemolithotroph)
Chemical energy source
(inorganic),Inorganic H/e-
donor,CO2 carbon source
Sulfur-oxdizing bacteria,
Hydrogen bacteria,
Nitrifying bacteria
Chemoheterotroph
(Chemoorganotroph)
Chemical energy source
(organic),Organic H/e-
donor,Organic carbon
source
Most bacteria,fungi,
protozoa
Nutritional types of microorganisms
Nutrient molecules frequently cannot cross
selectively permeable plasma membranes through
passive diffusion and must be transported by one of
three major mechanisms involving the use of
membrane carrier proteins.
Uptake of nutrients
Types of media
? Selective media,favor the growth of particular
microorganisms.
“加抑”, add componments which inhibite the
growth of unfavored bacteria.
“加富”, add components which promote the
growth of favored bacteria.
? Differential media,distinguish between different
groups of bacteria.
MacConkey agar,lactose,neutral red dye; lactose-
fermenting colonies appear pink to red in color.
Chapter4
Microbial growth
Isolation of pure cultures
the growth curve
Measurement of microbial growth
The continuous culture of microorganisms
Isolation of pure cultures
? Pure culture,a population of cells arising from a single cell,
to characterize an individual species,They are required
for the careful study of an individual microbial species.
? Single colony
? How to get
1,Dilute-pour/spread plate technique:
dilute?pour/spread ?isolated colonies
2,Streak plates,inoculating loop
Colony morphology and growth
individual species often form colonies of characteristic
size and appearance.
Microbial growth
? Batch culture,When microorganisms are
grown in a closed system,population
growth remains exponential for only a few
generations and then enters a stationary
phase due to factors like nutrient limitation
and waste accumulation.
? Continuous Culture,If a population is
cultured in an open system with continual
nutrient addition and waste removal,the
exponential phase can be maintained for
long periods,
.
The growth curve
The logarithm of the number of viable cells
versus the incubation time,
Generation time
? Example
100 bacteria present at time 0
If generation time is 2 hr
After 8 hr mass = 100 x 24
Direct counting
?Electronic counters,the Coulter Counter
for larger microorganisms such as protozoa,
algae,and nonfilamentous yeasts.
?The counting chamber
Disadvantages,1,The microbial population
must be fairly large for accuracy because such
a small volume is sampled.
2,Difficult to distinguish between living and
dead cells
Viable count,Spread plate method
and Pour plate method
Three basic steps,
? Dilution,
? Plating
? Incubation
Problems:
1,Clumps of cells.
colony forming units(CFU);
2,employed agar medium
cannot support growth of all
the viable microorganisms
present.
3,Hot agar,
spread plates>pour plates
The membrane filtration procedure
? Analyze aquatic samples
实验 9：滤膜法测定水中大肠菌群数（设计性）
Measuring bacterial mass (live + dead)
in liquid culture
Turbidity
(Cloudiness)
? The chemostat:
Chemostat used for continuous cultures,
rate of growth can be controlled either by
controlling the rate at which new medium
enters the growth chamber or by limiting a
required growth factor in the medium
? The turbidostat:
measures the absorbance or turbidity of the
culture in the growth vessel.
automatically regulated to maintain a
predetermined turbidity or cell density.
Chapter4
Microbial growth
Environmental factors on growth
Growth in natural environment
Water availability,pH,temperature,oxygen
concentration,pressure,radiation,and a
number of other environmental factors
influence microbial growth,
Yet many microorganisms,and particularly
bacteria,have managed to adapt and
flourish under environmental extremes that
would destroy most organisms.
Effect of environmental factors on growth
Hyperthermophiles have cell
membrane as lipid monolayer
Bacteria Archaea
? Microorganisms frequently change the pH
of their own habitat by producing acidic or
basic metabolic waste products.
? Buffers in media:
1,H2PO4-/HPO42-
2,CaCO3
3,Peptides and amino acids
Effects of Oxygen on Cell Growth
Terms used to describe O2 relationships of microorganisms:
? Aerobes:
– Obligate aerobes,oxygen presence necessary
– Facultative aerobes,oxygen not necessary,but
better with it
– Microaerophilic aerobes,required O2 at low level
than atmospheric
? Anaerobes,
– Aerotolerant,O2 not required,and growth no
better if O2 present
– Obligate (strict) anaerobes,Oxygen harmful or
lethal
Aerobic,anaerobic,facultative,micro-
aerophilic and aerotolerant anaerobe growth
A small amount of agar has
been added to keep the
liquid from becoming
disturbed and the redox dye,
resazurin,which is pink
when oxidized and colorless
when reduced,is added as a
redox indicator,
The above results were
obtained after a reducing
agent such as thioglycolate
(巯基醋酸盐 ) was added to
the medium.
Resazurin (刃天青 ),a redox
indicator dye,can be used to
detect oxygen in the
medium.
Different features
(a) Obligate aerobes-growth occurs only in the short
distance to which the oxygen diffuses into the medium,
(b) Facultative anaerobes growth is best near the surface,
where oxygen is available,but occurs throughout the tube,
(c) Obligate anaerobes-oxygen is toxic,and there is no
growth near the surface,
(d) Aerotolerant anaerobes-growth occurs evenly
throughout the tube but is not better at the surface because
the organisms do not use oxygen,
(e) Microaerophiles,aerobic organisms that do not tolerate
atmospheric concentrations of oxygen-growth occurs only
in a narrow band of optimal oxygen concentration.
How to culture
? Aerobic,culture vessel is shaken to aerate the medium
or sterile air must be pumped through the culture vessel.
? Anaerobes,
1,Media contain reducing agents such as
thioglycollate or crysteine, Boiling
2,Oxygen also may be eliminated from an anaerobic
system by removed air with a vacuum pump and
flushing out residual O2 with nitrogen gas.
3,GasPak jar,by using hydrogen and a palladium(钯 )
catalyst to remove O2 through the formatin of water.
4,Plastic bags or pouchs,only a few samples pouch,
catalyst + calcium carbonate?CO2
Toxic Forms of Oxygen
protein inactivation
?Singlet oxygen
?Superoxide anion (O2-)
? Hydrogen peroxide (H2O2)
? Hydroxyl radical (OH.)
Chapter 5
Microbial growth control
Physical methods
Chemical agents
Food Preservation
Conditions influencing the effectiveness of
antimicrobial agent activity
? 1,Population size.
? 2,Population composition,Spore,young/mature
? 3,Concentration or intensity of an antimicrobial agent.
? 4,Duration of exposure.
? 5,Temperature,higher
? 6,local environment.
eg,1,Heat kill more readily at an acid pH.
2,Organic matter protect microorganisms.
3,Biofilm protection.
Microbial Growth Control
? Physical ways
– Heat sterilization (including autoclave
and pasteurization)
– Radiation (microwaves,UV,X-rays,
g-rays and electrons)
– Filtration (depth filter,membrane
filter and nucleation track
(nucleopore) filter)
Evaluation of antimicrobial agent
effectiveness
? The phenol coefficient test:
? The test bacteria,Salmonella typhi
Staphylococcus aureus
a series of dilutions of phenol and the experimental
disinfectant.
bacteria + agent; 5min,10min,15min; subculture to
fresh medium,respectively; incubate 2-3 days.
The highest dilutions that kill the bacteria after a 10mins
exposure,but not after 5mins,are used to calculate the
phenol coefficient.
phenol coefficient =disinfect max dilution/phenol max
dilution
If >1,more effective than phenol
Measuring antimicrobial activity
Minimum inhibitory concentration (MIC)
Tube dilution technique
Agar diffusion method
6.1 General Properties of Viruses
6.2 Structure of viruses
6.3 The cultivation of viruses
6.4 General Features of Virus Reproduction
6.5 one-step growth curve
6.6 Temperate Bacteriophages,Lysogeny and Lambda
6.7 viral classification
6.8 Overview of Animal Viruses
6.9 plant viruses
6.10 Viroids and Prions
Chapter 6 Viruses
(1) their simple,acellular organization
(2) the absence of both DNA and RNA in the same
virion,
(3) their inability to reproduce independently of
cells and carry out cell division as prokaryotes
and eukaryotes do.
Viruses differ from living cells in at least three ways:
6.1 General Properties of Viruses
? Most viruses are too small to be seen under light microscope.
? All viruses consists of an RNA or DNA core genome
surrounded by a protein coat capsid.
? The combined viral genome and capsid is called the
nucleocapsid.
6.2 Structure of viruses
The nucleic acid of a virus is surrounded by a protein
coat called the capsid
Each capsid is composed of protein subunits called
capsomeres.
In some viruses,the capsid is covered by an envelope,
which usually consists of some combination of lipids,
proteins,and carbohydrates.
Depending on the virus,envelopes may or may not be
covered by spikes,which are carbohydrateprotein
complexes that project from the surface of the envelope.
A,Some viruses,such as tobacco mosaic
virus,have a helical symmetry with the
capsid surrounding an RNA genome.
B,Many viruses that infect bacteria,such as
the T-even bacteriophage,have a complex
capsid with DNA contained within the head
structure.
C,Some animal viruses,such as adenovirus,
have isometric symmetry and a DNA
genome.
D,others,such as coronavirus(冠状病毒 ）,
have complex capsids and an envelope with
protruding proteins surrounding an RNA
genome.
6.3 Cultivating Viruses
? Bacteriophage,bacterial Cultures
? Animal viruses,Tissue or cell cultures
? Plant viruses,tissue or separated cell
cultures,or cultures of protoplasts
Quantification
of viruses
? Plaque assay technique
for quantification of
bacterial viruses.
1,Attachment (adsorption) of
the virion to a susceptible host
cell.
2,Penetration (injection) of
the virion or its nucleic acid
into the cell.
7,Release of mature virions from the cell
6,Assembly of structural subunits (and membrane
components in enveloped viruses) and packaging of
nucleic acid into new virus particles.
5,Synthesis of proteins used as structural
subunits of the virus coat.
4,Replication of the virus nucleic acid.
3,Early steps in replication during which the host cell
biosynthetic machinery is altered as a prelude to virus
nucleic acid synthesis,Virus-specific enzymes are typically
made.
1,Latent period,no
release of virions
2,Rise period or
burst,the host
cells rapidly lyse
and release
infective phages
The one-step growth curve of virus replication
The consequences of
infection by a temperate
bacteriophage,The
alternatives on infection
are integration of the
virus DNA into the host
DNA (lysogenization) or
replication and release
of mature virus (lysis),
The lysogenic cell can
also be induced to
produce mature virus
and lyse,
6.10 Viroids and Prions
Viroids are small,circular,single-stranded RNA
molecules that are the smallest known pathogens,
? about 250 to 370 nucleotides long,
? Viroids are found principally in the nucleolus of
infected cells; 200-2000 copies
? They do not act as mRNAs to direct protein
synthesis,How to cause disease symptoms
? Could be replicated by an RNA-dependent
RNA polymerase,a rolling-circle type
mechanism
Chapter7 Microbial metablism
Catabolism,anabolism
Fermentation
Respiration
Nitrogen fixtation
The synthesis of peptiglydogen
Three type fermentation of
Saccharomyces cerevisiae
I,pyruvate?acetaldehyde?ethanol
II,pH7,glycerol
III,NaHSO3
Eight stages of Peptidoglycan
synthesis
1,The formation of UDP-NAM and UDP-NAG
2,Amino acids are sequentially added to UDP-
NAM to form the pentapeptide chain.
3,The NAM-pentapeptide is transferred from
UDP to a bactoprenol phosphate at the
membrane surfacre.
4,UDP-NAG adds NAG to the NAM-
pentapeptide to form the peptidoglycan repeat
unit.
5,Repeat unit is transported across the
membrane to its outer surface by the
bactoprenol pyrophosphate carrier.
6,The peptidoglycan unit is attached to the
growing end of a peptidoglycan chain to
lengthen it by one repeat unit.
7,The bactoprenol carrier returns to the inside
of the membrane,A phosphate is released.
8,Peptide cross-links between the peptidoglycan
chains are formed by transpeptidation.
Eight stages of Peptidoglycan
synthesis
Nitrogen fixation
The reduction of atmospheric gaseous nitrogen
to ammonia is callled nitrogen fixation.
Nitrogen fixation occurs in:
1,Free-living bacteria.(Azotobacter)
2,Bacteria living in symbiotic association with
plants such as legumes(Rhizobium)
3,cyanobacteria
Chapte8 Microbial genetics
8.1 DNA as genetic material
8.2 Mutation
8.3 DNA repair
? Transition mutations,purine for
putine or pyrimidine for pyrimidine
substitutions.
relatively common
? Transversion mutations,a purine is
substituted for a pyrimidine,or a
pyrimidine for a purine.
rarer
Molecular
basis of
Mutation
? Point Mutation(one
base pair mutant)
– Silent mutation
– Nonsense mutation
– Missense
mutation(the effect
may range from
complete loss of
activity to no change
at all)
天
冬
酰
胺
酪
氨
酸
Frameshift mutations
? Occur in protein-encoding gene that including
the reading frame
? If occurs in promoter region,it is not frameshift
Back Mutations or Reversions
? Point Mutations are reversible
? Revertant
– wild type phenotype that was lost in the
mutant is restored.
? Same site revertants
? True revertants
? Second site mutation
– Suppressor mutation,restore the wild type
phenotype by mutation somewhere else.
Isolation of Mutants
? Pigmented and
nonpigmented
mutants
? Antibiotic-resistant
mutants within the
inhibition zone
? Genetic marker,
such as the
expression of b-
galactosidase that
produces the blue
color
Mutants that are detectable
? Non-motile
? Non-capsulated
? Rough colony
? Nutritional
? Sugar fermentation
? Drug resistant
? Virus resistant
? Temperature sensitive
? Pigmentless
? Cold sensitive
Mutagenesis and Carcinogenesis
The Ames Test
? Bacteria can be used as screening agents for
the potential mutagenicity of chemicals
– As it has been found that many mutagenic chemicals are
also carcinogenic,capable of causing cancer in animals and
humans
? The fact that a compound is mutagenic in a bacterial
system serves as a warning of possible danger
? Dr,Bruce Ames at the UC Berkeley has developed
the Ames Test technique for the screening of
carcinogenic compounds.
8.3 DNA repair
? 1,Excision repair,first excise,then DNA
polymerase I,at last DNA ligase join
? 2,Removal of lesions (photoreactivation)
splitting thymine dimers apart into separate
thymines with the help of visible light.
? 3,Postreplication repair
mismatch repair system,excise ? replace ? seal
? 4,Recombination repair,recA protein
? 5,SOS repair
Plasmid
? Conjugative,plasmids which govern their own transfer by
cell-to-cell contact are called conjugative
? Tra region,a set of genes within the plasmid that control the
transmissability by conjugation
? Hfr (high frequency of recombination),strains of bacteria
that transfer large amounts of chromosomal DNA during
conjugation
? Supercoil,plasmids isolated from the cells are in supercoiled
configuration
? Plasmid separation,by ultracentrifugation or electrophoresis
? Curing of plasmids,elimination of plasmids from host cells
by various treatments.
(1) Transformation,which involves donor DNA free
in the environment
(2) Transduction,in which the donor DNA transfer
is mediated by a virus
(3) Conjugation,in which the transfer involves cell-
to-cell contact and a conjugative plasmid in the donor
cell
8.5 Three main processes of genetic recombination in
prokaryotes fragments of homologous DNA from a donor
chromosome are transferred to a recipient cell
Three types of transposable elements in bacteria
? Insertion sequences (IS),about 1000 nucleotides,
carry only information to move them to new
location (IS1,IS2 and IS3).
? Transposons (Tn),larger than IS,carry genes,
such as drug resistance markers and other
selectable genes.
? Some special viruses (such as Mu)
Three separate but interrelated parts,
1,Classification,the arrangement of organisms
into groups or taxa based on mutual similarity
or evolutionary relatedness.
2,Nomenclature,the branch of taxonomy
concerned with the assignment of names to
taxonomic groups in agreement with
published rules.
3,Identification,the practical side of taxonomy,
the process of determining that a particular
isolate belongs to a recognized taxon.
Chapter9 Microbial taxonomy
Ribosomal RNAs as Evolutionary Chronometers
? Reasons:
– Ancient molecules
– Functionally constant
– Universally distributed
– Moderately well conserved in sequence
across broad phylogenetic distances
,official” taxonomy and
nomenclature
? Taxonomy,Bergey’s manual of systematic
bacteriology or Bergey’s manual of
determinative bacteriology
in some sence official
? Nomenclature,all new names are validly
published to gain standing in the
nomenclature,either by being published in
papers in the international Journal of
Systematic Bacteriology or,if published
elsewhere,by being announced in the
Validation Lists
10.1 Microorganisms in nature ecosystem
10.2 Microbial population interactions
10.3 Biogeochemical cycles
10.4 Plant-microbe interactions
10.5 Bioremediation
Chapter 10 Microbial Ecology
The rhizosphere is the soil region
in close contact with plant roots.
Within the rhizosphere,the plant roots
exert a direct influence on the soil bacteria,
This influence is known as the
rhizosphere effect,
In the rhizosphere,microbial populations reach much
higher densities in the rhizosphere than in the free soil.
Rhizosphere Effect
( R/S ratio )
1,neutralism (中性关系 )
2,commensalism (偏利关系 )
3,synergism (协同关系 )
4,mutualism (互惠关系 )
5,competition (竞争关系 )
6,antagonism (拮 抗关系 )
7,parasitism (寄生关系 )
8,predation (捕食关系 )
10.2 Microbial population interactions
10.3 Biogeochemical cycles
1,Carbon cycle
2,Nitrogen cycle
3,Sulfur cycle
4,Iron cycle
Measuring water quality
TOC:total organic carbon
COD:chemical oxygen demand
BOD:biochemical oxygen demand(20 ℃ 5days）
Important Persons in Microbiology History
1684 Antonie van Leeuwenhoek (discovery of bacteria)
1857-1864 Louis Pasteur (lactic acid fermentation,yeast
alcohol fermentation,spontaneous generation theory
“swan neck flasks”)
1881-1884 Robert Koch (pure culture,cause of
tuberculosis,Koch’s postulates,cause of (霍乱 )
cholera）
1889 Martinus Beijerinck (concept of a virus)
1929 Alexander Fleming (discovery of penicilin)
1953 James Watson,Francis Crick (DNA structure)
1983 Luc Montagnier (discovery of HIV)
Prokaryotic Cell (versus
Eukaryotic Cell)
? Not compartmentalized
? Cell membranes lack sterols (e.g,cholesterol)
? Single circular chromosome
? Ribosomal are 70S with
– subunits 30S (16S rRNA)
– 50S (5S & 23S rRNA)
Coccus or Cocci
Rod
Spirillum or Spirilla
Spirochete
Budding and appendaged
Filamentous
2.Shape and arrangement
Procaryoic cell organization
Mesosome,
invaginationa of the plasma membrane
in the shape of vesicles,tubules,or lamellae.
Functions:
1.cell wall formation during division.
2.play a role in chromosome replicaton
and distribution to daughter cells.
Making Wall-less forms
? Result from action of:
–enzymes lytic for cell wall
–antibiotics inhibiting peptidoglycan
biosynthesis
? Wall-less bacteria that don’t replicate:
–spheroplasts (with outer membrane)?
–protoplasts (no outer membrane),?
? Wall-less bacteria that replicate
–L forms
Naturally Wall-less Genus
Mycloplasma
Within the cytoplasm of procaryotic (and eucaryotic)
cells are several kinds of reserve deposits,known as
inclusions,Some inclusions are common to a wide
variety of bacteria,whereas others are limited to a
small number of species and therefore serve as a
basis for identification,Among the more prominent
bacterial inclusions are the following:
Carbon storage polymers – PHB and glycogen
Phosphate polymers
Sulfur Granules
Gas Vacuoles
INCLUSIONS
Peptidoglycan
Muramic acid
Glucosamine
L-alanine
D-glutamic acid
L-lysine/Diaminopimelic acid
D-alanine
D-alanine
The mechanism of Gram staining
During the procedure the bacteria are first stained with
crystal violet and next treated with iodine to promote dye
retention,When gram-positive bacteria then are decolorized
with ethanol,the alcohol is thought to shrink the pores of the
thick peptidoglycan,Thus the dye-iodine complex is retained
during the short decolorization step and the bacteria remain
purple,In contrast,gram-negative bacteria peptidoglycan is
very thin,not as highly cross-linked,and has larger pores,
Alcohol treatment also may extract enough lipid from the
gram-neaative wall to increase its porosity further,For these
reasons,alcohol more readily removes the purple crystal
violet-iodine complex from gram-negative bacteria.
Special structure
? Pili and fimbriae
? Flagella
? The bacterial endospore
Chapter 1
The structure and function of prokaryotes
Pili and fimbriae
G-,short,fine,hairlike appendages
that are not involved in motility.
Fimbriae/fimbria:1000piece/cell,
adhesion to host epithelium
Sex pili/pilus,1-10piece/cell,
sexual conjugation,they are
genetically determined by sex factors
or conjugative plasmids and are required
for bacterial mating,
Receptors of some bacterial viruses.
Flagellar Arrangements
Amphitrichous
Peritrichous
Polar/monotrichous
Lophotrichous
Flagellar ultrastructure
Filament,from cell surface to the tip
Basal body,embedded in the cell
Hook,link the filament to its basal body
Basal body,
G+,
S (peptidoglycan),
M(plasma membrane)
G-,
L (out membrane),
P (peptidoglycan layer),
S-M(contact the plasma membrane)
The E,coli
Flagellar
Structure
Bacterial Behavior
Chemotaxis,Phototaxis,and Other Taxes
? Prokaryotes encounter gradients of physical
and chemical agents in nature,and the motility
machinery in the cell is designed to respond in
a positive or negative way to these gradients
by directing the movement of the cell either
toward or away from the signal molecule,
respectively.
? Such directed movements are called TAXES.
? Chemotaxis,a response to chemicals.
? Phototaxis,a response to light.
How to judge wether a cell
have flagella
? 1,Light Microscope by staining
? 2,Electron microscope
? 3,Feature of colony
? 4,Culture in semi-solid medium
Endospore Structure
Mature free endospore
Spore formation,lack of nutrients,7 stages
The mechnism of heat and other
kethal agents resistantance
1,Calcium dipicolinic acid
2,Heat stable enzyme
3,High G+C%
4,Less water
5,Amino acid containing surfur(cys)
Paraspore crystal as biopesticide
Importance of fungi
? 1,decomposers
? 2,Major cause of plant diseases
? 3,Many diseases of animals and humans are caused by
fungi
? 4,products,the making of bread,wine,beer(yeasts);
the preparation of some cheeses,soy sause and sufu; the
commercial production
of many organic acids and certain drugs; The
manufacture of many antibiotics and
immunosuppressive drug cyclosporine.
? 5,Important research tools in the study of fundamental
biological processes.
YM shift
? Many fungi that cause diseases in humans and
animals,are dimorphic,they have two forms.
? 1,The yeast (Y) form in the animals
? 2,The mold or mycelial form (M) in the external
enviroment.
? Plant-associated fungi,the mycelial form in the
plantl the yeast form in the external enviroment.
yeasts
? Unicellular fungus,spherical to egg
shaped,single nucleus and
repriduces either asexually by
budding and transverse division or
sexually throough spore formation
? No flagella,possess most of the other
eucaryotic organelles..
Cilia and flagella structure
? Fig4.24p90
Chapter3
Microbial nutrition
1.Nutrient requirement
2.Nutritional types of microorganisms
3.Uptake of nutrients
4.Culture media
Growth Factors
Amino acids are needed for protein synthesis,
purines and pyrimidines for nucleic acid synthesis,
Vitamins are small organic molecules that usually
make up all or part enzyme cofactors,and only
very small amounts are required for growth,
(1)amino acids,(2) purines and pyrimidines,(3) vitamins
Major nutritional
type
Sources of energy,
hydrogen/electrons,
and carbon
Representative
microorganisms
Photoautotroph
(Photolithotroph)
Light energy,inorganic
hydrogen/electron(H/e-)
donor,CO2 carbon source
Algae,Purple and green
bacteria,Cyanobacteria
Photoheterotroph
(Photoorganotroph)
Light energy,inorganic
H/e- donor,
Organic carbon source
Purple nonsulfur bacteria,
Green sulfur bacteria
Chemoautotroph
(Chemolithotroph)
Chemical energy source
(inorganic),Inorganic H/e-
donor,CO2 carbon source
Sulfur-oxdizing bacteria,
Hydrogen bacteria,
Nitrifying bacteria
Chemoheterotroph
(Chemoorganotroph)
Chemical energy source
(organic),Organic H/e-
donor,Organic carbon
source
Most bacteria,fungi,
protozoa
Nutritional types of microorganisms
Nutrient molecules frequently cannot cross
selectively permeable plasma membranes through
passive diffusion and must be transported by one of
three major mechanisms involving the use of
membrane carrier proteins.
Uptake of nutrients
Types of media
? Selective media,favor the growth of particular
microorganisms.
“加抑”, add componments which inhibite the
growth of unfavored bacteria.
“加富”, add components which promote the
growth of favored bacteria.
? Differential media,distinguish between different
groups of bacteria.
MacConkey agar,lactose,neutral red dye; lactose-
fermenting colonies appear pink to red in color.
Chapter4
Microbial growth
Isolation of pure cultures
the growth curve
Measurement of microbial growth
The continuous culture of microorganisms
Isolation of pure cultures
? Pure culture,a population of cells arising from a single cell,
to characterize an individual species,They are required
for the careful study of an individual microbial species.
? Single colony
? How to get
1,Dilute-pour/spread plate technique:
dilute?pour/spread ?isolated colonies
2,Streak plates,inoculating loop
Colony morphology and growth
individual species often form colonies of characteristic
size and appearance.
Microbial growth
? Batch culture,When microorganisms are
grown in a closed system,population
growth remains exponential for only a few
generations and then enters a stationary
phase due to factors like nutrient limitation
and waste accumulation.
? Continuous Culture,If a population is
cultured in an open system with continual
nutrient addition and waste removal,the
exponential phase can be maintained for
long periods,
.
The growth curve
The logarithm of the number of viable cells
versus the incubation time,
Generation time
? Example
100 bacteria present at time 0
If generation time is 2 hr
After 8 hr mass = 100 x 24
Direct counting
?Electronic counters,the Coulter Counter
for larger microorganisms such as protozoa,
algae,and nonfilamentous yeasts.
?The counting chamber
Disadvantages,1,The microbial population
must be fairly large for accuracy because such
a small volume is sampled.
2,Difficult to distinguish between living and
dead cells
Viable count,Spread plate method
and Pour plate method
Three basic steps,
? Dilution,
? Plating
? Incubation
Problems:
1,Clumps of cells.
colony forming units(CFU);
2,employed agar medium
cannot support growth of all
the viable microorganisms
present.
3,Hot agar,
spread plates>pour plates
The membrane filtration procedure
? Analyze aquatic samples
实验 9：滤膜法测定水中大肠菌群数（设计性）
Measuring bacterial mass (live + dead)
in liquid culture
Turbidity
(Cloudiness)
? The chemostat:
Chemostat used for continuous cultures,
rate of growth can be controlled either by
controlling the rate at which new medium
enters the growth chamber or by limiting a
required growth factor in the medium
? The turbidostat:
measures the absorbance or turbidity of the
culture in the growth vessel.
automatically regulated to maintain a
predetermined turbidity or cell density.
Chapter4
Microbial growth
Environmental factors on growth
Growth in natural environment
Water availability,pH,temperature,oxygen
concentration,pressure,radiation,and a
number of other environmental factors
influence microbial growth,
Yet many microorganisms,and particularly
bacteria,have managed to adapt and
flourish under environmental extremes that
would destroy most organisms.
Effect of environmental factors on growth
Hyperthermophiles have cell
membrane as lipid monolayer
Bacteria Archaea
? Microorganisms frequently change the pH
of their own habitat by producing acidic or
basic metabolic waste products.
? Buffers in media:
1,H2PO4-/HPO42-
2,CaCO3
3,Peptides and amino acids
Effects of Oxygen on Cell Growth
Terms used to describe O2 relationships of microorganisms:
? Aerobes:
– Obligate aerobes,oxygen presence necessary
– Facultative aerobes,oxygen not necessary,but
better with it
– Microaerophilic aerobes,required O2 at low level
than atmospheric
? Anaerobes,
– Aerotolerant,O2 not required,and growth no
better if O2 present
– Obligate (strict) anaerobes,Oxygen harmful or
lethal
Aerobic,anaerobic,facultative,micro-
aerophilic and aerotolerant anaerobe growth
A small amount of agar has
been added to keep the
liquid from becoming
disturbed and the redox dye,
resazurin,which is pink
when oxidized and colorless
when reduced,is added as a
redox indicator,
The above results were
obtained after a reducing
agent such as thioglycolate
(巯基醋酸盐 ) was added to
the medium.
Resazurin (刃天青 ),a redox
indicator dye,can be used to
detect oxygen in the
medium.
Different features
(a) Obligate aerobes-growth occurs only in the short
distance to which the oxygen diffuses into the medium,
(b) Facultative anaerobes growth is best near the surface,
where oxygen is available,but occurs throughout the tube,
(c) Obligate anaerobes-oxygen is toxic,and there is no
growth near the surface,
(d) Aerotolerant anaerobes-growth occurs evenly
throughout the tube but is not better at the surface because
the organisms do not use oxygen,
(e) Microaerophiles,aerobic organisms that do not tolerate
atmospheric concentrations of oxygen-growth occurs only
in a narrow band of optimal oxygen concentration.
How to culture
? Aerobic,culture vessel is shaken to aerate the medium
or sterile air must be pumped through the culture vessel.
? Anaerobes,
1,Media contain reducing agents such as
thioglycollate or crysteine, Boiling
2,Oxygen also may be eliminated from an anaerobic
system by removed air with a vacuum pump and
flushing out residual O2 with nitrogen gas.
3,GasPak jar,by using hydrogen and a palladium(钯 )
catalyst to remove O2 through the formatin of water.
4,Plastic bags or pouchs,only a few samples pouch,
catalyst + calcium carbonate?CO2
Toxic Forms of Oxygen
protein inactivation
?Singlet oxygen
?Superoxide anion (O2-)
? Hydrogen peroxide (H2O2)
? Hydroxyl radical (OH.)
Chapter 5
Microbial growth control
Physical methods
Chemical agents
Food Preservation
Conditions influencing the effectiveness of
antimicrobial agent activity
? 1,Population size.
? 2,Population composition,Spore,young/mature
? 3,Concentration or intensity of an antimicrobial agent.
? 4,Duration of exposure.
? 5,Temperature,higher
? 6,local environment.
eg,1,Heat kill more readily at an acid pH.
2,Organic matter protect microorganisms.
3,Biofilm protection.
Microbial Growth Control
? Physical ways
– Heat sterilization (including autoclave
and pasteurization)
– Radiation (microwaves,UV,X-rays,
g-rays and electrons)
– Filtration (depth filter,membrane
filter and nucleation track
(nucleopore) filter)
Evaluation of antimicrobial agent
effectiveness
? The phenol coefficient test:
? The test bacteria,Salmonella typhi
Staphylococcus aureus
a series of dilutions of phenol and the experimental
disinfectant.
bacteria + agent; 5min,10min,15min; subculture to
fresh medium,respectively; incubate 2-3 days.
The highest dilutions that kill the bacteria after a 10mins
exposure,but not after 5mins,are used to calculate the
phenol coefficient.
phenol coefficient =disinfect max dilution/phenol max
dilution
If >1,more effective than phenol
Measuring antimicrobial activity
Minimum inhibitory concentration (MIC)
Tube dilution technique
Agar diffusion method
6.1 General Properties of Viruses
6.2 Structure of viruses
6.3 The cultivation of viruses
6.4 General Features of Virus Reproduction
6.5 one-step growth curve
6.6 Temperate Bacteriophages,Lysogeny and Lambda
6.7 viral classification
6.8 Overview of Animal Viruses
6.9 plant viruses
6.10 Viroids and Prions
Chapter 6 Viruses
(1) their simple,acellular organization
(2) the absence of both DNA and RNA in the same
virion,
(3) their inability to reproduce independently of
cells and carry out cell division as prokaryotes
and eukaryotes do.
Viruses differ from living cells in at least three ways:
6.1 General Properties of Viruses
? Most viruses are too small to be seen under light microscope.
? All viruses consists of an RNA or DNA core genome
surrounded by a protein coat capsid.
? The combined viral genome and capsid is called the
nucleocapsid.
6.2 Structure of viruses
The nucleic acid of a virus is surrounded by a protein
coat called the capsid
Each capsid is composed of protein subunits called
capsomeres.
In some viruses,the capsid is covered by an envelope,
which usually consists of some combination of lipids,
proteins,and carbohydrates.
Depending on the virus,envelopes may or may not be
covered by spikes,which are carbohydrateprotein
complexes that project from the surface of the envelope.
A,Some viruses,such as tobacco mosaic
virus,have a helical symmetry with the
capsid surrounding an RNA genome.
B,Many viruses that infect bacteria,such as
the T-even bacteriophage,have a complex
capsid with DNA contained within the head
structure.
C,Some animal viruses,such as adenovirus,
have isometric symmetry and a DNA
genome.
D,others,such as coronavirus(冠状病毒 ）,
have complex capsids and an envelope with
protruding proteins surrounding an RNA
genome.
6.3 Cultivating Viruses
? Bacteriophage,bacterial Cultures
? Animal viruses,Tissue or cell cultures
? Plant viruses,tissue or separated cell
cultures,or cultures of protoplasts
Quantification
of viruses
? Plaque assay technique
for quantification of
bacterial viruses.
1,Attachment (adsorption) of
the virion to a susceptible host
cell.
2,Penetration (injection) of
the virion or its nucleic acid
into the cell.
7,Release of mature virions from the cell
6,Assembly of structural subunits (and membrane
components in enveloped viruses) and packaging of
nucleic acid into new virus particles.
5,Synthesis of proteins used as structural
subunits of the virus coat.
4,Replication of the virus nucleic acid.
3,Early steps in replication during which the host cell
biosynthetic machinery is altered as a prelude to virus
nucleic acid synthesis,Virus-specific enzymes are typically
made.
1,Latent period,no
release of virions
2,Rise period or
burst,the host
cells rapidly lyse
and release
infective phages
The one-step growth curve of virus replication
The consequences of
infection by a temperate
bacteriophage,The
alternatives on infection
are integration of the
virus DNA into the host
DNA (lysogenization) or
replication and release
of mature virus (lysis),
The lysogenic cell can
also be induced to
produce mature virus
and lyse,
6.10 Viroids and Prions
Viroids are small,circular,single-stranded RNA
molecules that are the smallest known pathogens,
? about 250 to 370 nucleotides long,
? Viroids are found principally in the nucleolus of
infected cells; 200-2000 copies
? They do not act as mRNAs to direct protein
synthesis,How to cause disease symptoms
? Could be replicated by an RNA-dependent
RNA polymerase,a rolling-circle type
mechanism
Chapter7 Microbial metablism
Catabolism,anabolism
Fermentation
Respiration
Nitrogen fixtation
The synthesis of peptiglydogen
Three type fermentation of
Saccharomyces cerevisiae
I,pyruvate?acetaldehyde?ethanol
II,pH7,glycerol
III,NaHSO3
Eight stages of Peptidoglycan
synthesis
1,The formation of UDP-NAM and UDP-NAG
2,Amino acids are sequentially added to UDP-
NAM to form the pentapeptide chain.
3,The NAM-pentapeptide is transferred from
UDP to a bactoprenol phosphate at the
membrane surfacre.
4,UDP-NAG adds NAG to the NAM-
pentapeptide to form the peptidoglycan repeat
unit.
5,Repeat unit is transported across the
membrane to its outer surface by the
bactoprenol pyrophosphate carrier.
6,The peptidoglycan unit is attached to the
growing end of a peptidoglycan chain to
lengthen it by one repeat unit.
7,The bactoprenol carrier returns to the inside
of the membrane,A phosphate is released.
8,Peptide cross-links between the peptidoglycan
chains are formed by transpeptidation.
Eight stages of Peptidoglycan
synthesis
Nitrogen fixation
The reduction of atmospheric gaseous nitrogen
to ammonia is callled nitrogen fixation.
Nitrogen fixation occurs in:
1,Free-living bacteria.(Azotobacter)
2,Bacteria living in symbiotic association with
plants such as legumes(Rhizobium)
3,cyanobacteria
Chapte8 Microbial genetics
8.1 DNA as genetic material
8.2 Mutation
8.3 DNA repair
? Transition mutations,purine for
putine or pyrimidine for pyrimidine
substitutions.
relatively common
? Transversion mutations,a purine is
substituted for a pyrimidine,or a
pyrimidine for a purine.
rarer
Molecular
basis of
Mutation
? Point Mutation(one
base pair mutant)
– Silent mutation
– Nonsense mutation
– Missense
mutation(the effect
may range from
complete loss of
activity to no change
at all)
天
冬
酰
胺
酪
氨
酸
Frameshift mutations
? Occur in protein-encoding gene that including
the reading frame
? If occurs in promoter region,it is not frameshift
Back Mutations or Reversions
? Point Mutations are reversible
? Revertant
– wild type phenotype that was lost in the
mutant is restored.
? Same site revertants
? True revertants
? Second site mutation
– Suppressor mutation,restore the wild type
phenotype by mutation somewhere else.
Isolation of Mutants
? Pigmented and
nonpigmented
mutants
? Antibiotic-resistant
mutants within the
inhibition zone
? Genetic marker,
such as the
expression of b-
galactosidase that
produces the blue
color
Mutants that are detectable
? Non-motile
? Non-capsulated
? Rough colony
? Nutritional
? Sugar fermentation
? Drug resistant
? Virus resistant
? Temperature sensitive
? Pigmentless
? Cold sensitive
Mutagenesis and Carcinogenesis
The Ames Test
? Bacteria can be used as screening agents for
the potential mutagenicity of chemicals
– As it has been found that many mutagenic chemicals are
also carcinogenic,capable of causing cancer in animals and
humans
? The fact that a compound is mutagenic in a bacterial
system serves as a warning of possible danger
? Dr,Bruce Ames at the UC Berkeley has developed
the Ames Test technique for the screening of
carcinogenic compounds.
8.3 DNA repair
? 1,Excision repair,first excise,then DNA
polymerase I,at last DNA ligase join
? 2,Removal of lesions (photoreactivation)
splitting thymine dimers apart into separate
thymines with the help of visible light.
? 3,Postreplication repair
mismatch repair system,excise ? replace ? seal
? 4,Recombination repair,recA protein
? 5,SOS repair
Plasmid
? Conjugative,plasmids which govern their own transfer by
cell-to-cell contact are called conjugative
? Tra region,a set of genes within the plasmid that control the
transmissability by conjugation
? Hfr (high frequency of recombination),strains of bacteria
that transfer large amounts of chromosomal DNA during
conjugation
? Supercoil,plasmids isolated from the cells are in supercoiled
configuration
? Plasmid separation,by ultracentrifugation or electrophoresis
? Curing of plasmids,elimination of plasmids from host cells
by various treatments.
(1) Transformation,which involves donor DNA free
in the environment
(2) Transduction,in which the donor DNA transfer
is mediated by a virus
(3) Conjugation,in which the transfer involves cell-
to-cell contact and a conjugative plasmid in the donor
cell
8.5 Three main processes of genetic recombination in
prokaryotes fragments of homologous DNA from a donor
chromosome are transferred to a recipient cell
Three types of transposable elements in bacteria
? Insertion sequences (IS),about 1000 nucleotides,
carry only information to move them to new
location (IS1,IS2 and IS3).
? Transposons (Tn),larger than IS,carry genes,
such as drug resistance markers and other
selectable genes.
? Some special viruses (such as Mu)
Three separate but interrelated parts,
1,Classification,the arrangement of organisms
into groups or taxa based on mutual similarity
or evolutionary relatedness.
2,Nomenclature,the branch of taxonomy
concerned with the assignment of names to
taxonomic groups in agreement with
published rules.
3,Identification,the practical side of taxonomy,
the process of determining that a particular
isolate belongs to a recognized taxon.
Chapter9 Microbial taxonomy
Ribosomal RNAs as Evolutionary Chronometers
? Reasons:
– Ancient molecules
– Functionally constant
– Universally distributed
– Moderately well conserved in sequence
across broad phylogenetic distances
,official” taxonomy and
nomenclature
? Taxonomy,Bergey’s manual of systematic
bacteriology or Bergey’s manual of
determinative bacteriology
in some sence official
? Nomenclature,all new names are validly
published to gain standing in the
nomenclature,either by being published in
papers in the international Journal of
Systematic Bacteriology or,if published
elsewhere,by being announced in the
Validation Lists
10.1 Microorganisms in nature ecosystem
10.2 Microbial population interactions
10.3 Biogeochemical cycles
10.4 Plant-microbe interactions
10.5 Bioremediation
Chapter 10 Microbial Ecology
The rhizosphere is the soil region
in close contact with plant roots.
Within the rhizosphere,the plant roots
exert a direct influence on the soil bacteria,
This influence is known as the
rhizosphere effect,
In the rhizosphere,microbial populations reach much
higher densities in the rhizosphere than in the free soil.
Rhizosphere Effect
( R/S ratio )
1,neutralism (中性关系 )
2,commensalism (偏利关系 )
3,synergism (协同关系 )
4,mutualism (互惠关系 )
5,competition (竞争关系 )
6,antagonism (拮 抗关系 )
7,parasitism (寄生关系 )
8,predation (捕食关系 )
10.2 Microbial population interactions
10.3 Biogeochemical cycles
1,Carbon cycle
2,Nitrogen cycle
3,Sulfur cycle
4,Iron cycle
Measuring water quality
TOC:total organic carbon
COD:chemical oxygen demand
BOD:biochemical oxygen demand(20 ℃ 5days）
