Chapter 5 Microbial Nutrition
and Culture medium
Nutrition, Nutrition is a process by which chemical
substances called nutrients are required from the
environment and used for the cellular activities of
metabolism and growth,
Nutrient,the substance could be used by microbes for
metabolism or reproduction of cells,
Nutrient provide the structural substance,energy,
metabolic regulate substance and appropriate
physiological environment,
Some microbes could use nonmaterial energy---light,
I,cellular chemical constitutes
It’s much alike in the life in the element, The main elements
are C,H,O,N (90%---97% to total dry cell weight),C is
about 50%,the others are inorganic nutrient,C/N is 5:1
usually,
Section 1 Six kinds of essential
nutrient for microbes
element
?Macronutrient, C,H,O,N,P,S
?others element,K,Na,Ca,Mg,Fe,Mn、
Cu,Co,Zn,Mo,et al
Existence form,
? organic,protein,sugar,lipoid、
nucleic acid,vitamin,decomposition
substance,metabolic medial products
? inorganic salt ash
? Water---70% ~ 90% to wet cell
weight
element
bacteria
yeast mould
C 50
49.8
47.0
N
15
7.5
5.2
H
8
5.7
6.7
O
20
31.1
40.2
P
3
1.5
1.2
S
1
0.3
0.2
Table 1.The macronutrient content in microbes (%
to dry cell weight)
The substance utilized by generic species
could be utilized by microbes too,
The substance couldn’t be utilized by generic
species could be utilized by microbes,
Some kinds of substance harmful to generic
species also could be utilized by microbes,
Microbes is omnivorous,
II,Main nutrient and the function
Carbon source
Nitrogen source
Energy source
Growth factor
Inorganic salt
Water
Functions,
vconstitute the cells of the microbes
vprovide the energy needed in the
physiological activity to microbes
vthe source to form the microbial
metabolic products
Nutrient is the substance base for
microbial metabolism and living activity,
If lose this base,the life will die,
Heterotrophic microbe,is an organism must obtain its
carbon in an organic form,
Autotrophic microbe,is an organism that uses CO2,an
inorganic gas,as its carbon source,
1.Carbon sources
The nutrient provide the carbon element
for microbies
?Organic carbon sources,protein,nucleic
acid,starch,glucose,et al
?Inorganic acid,CO2,Na2CO3,CaCO3 et al
the carbon sources could be utilized by microbes
(compound)
saccharide,glucose,fructose,maltose,sucrose，
starch,galactose,lactose,mannancellulose，
hemicellulose,lignin,et al
organic acid,lactic acid,citric acid，,short chain
fatty acid,long chain fatty acid, amino acid,et al
alcoholic aldehyde,ethanol,et al
fat,fat,lecithoid,et al
hydrocarbon,natural gas,petroleum,olefin,et al
CO2
carbonate,NaHCO3,CaCO3,chalk,et al
others,cyanide,protein,peptide,nucleic acid
?organic nitrogen sources, peptone,soybean powder
,maize slurry
?inorganic nitrogen sources,NH4NO3,(NH4)2SO4
?gas nitrogen sources,N
2 in the atmosphere
2.Nitrogen sources
The nutrient provide the nitrogen element for microbes,
Normally,nitrogen source couldn’t be used as energy sources,
?quick nitrogen sources
?slow nitrogen sources
energy sources are the substances or radiant energy that could provide
primal energy sources, Heterotrophs’s carbon sources could also be its
energy sources,
3,Energy sources
Inorganic:energy to chemoautotrophs
Energy sources
table
Chemical substances
Radiant energy,energy to photoautotrophs and
photoheterotrophs,
Organic,energy to chemoheterotrophs
mono-function, radiant energy
?di-function,reductive inorganic nutrient,such as NH4+ is the
energy sources as well as the nitrogen sources of nitrate
bacterial,
?triple function,the nutrients including N, C, H, O are
energy sources, carbon sources and nitrogen sources
heterotrophs microbes,
one kind of nutrient could provide more than one kind of function
Definition:An organic compound such as an amino
acid,nitrogen base,or vitamin that cannot be
synthesized by an organism and must be provided as
a nutrient is a growth factor,
Sources of growth factors in culture medium,
yeast extract,maize slurry,wort,et al。
4,Growth factor
Function,coenzyme or necessary to activation of
enzyme,
narrow sense,vitamin
broad sense,vitamin,amino acid,base,fatty
acid,et al
?auxoautotrophs
?auxoheterotrophs
?nutritional deficiency mutant
?auxo-excessivetrophs
vitamin
species
thiamin B
1
Bacillus anthracis
lactoflavin
Clostridium tetani
nicotinic acid
Brucella abortus
vitamin B6
Lactobacillus spp,
biotin
Leuconostoc mesenteroides
pantothenic acid
Proteus morganii
folacin
Leuconostoc dextranicum
B12
Lactobacillus spp,
vitamin K
Bacteroides melaninogenicus
Vitamin necessary to some
bacterials
5,Inorganic salts
Macroelement,need the concentration of
the element at 10-3-10-4M
Microelement,need the concentration of
the element at 10-6-10-8M
The physiology function for inorganic salts
inorganic
salts
Macroelement
Microelement
Usual
function
Especial
function
Normal molecular in cell(P,S,Ca,Mg,Fe)
Physiology
modulation material
maintain osmotic pressure (Na+)
enzyme activator(Mg2+)
stablize pH
Energy sourse of chemoautotrophic bacteria (S,Fe2+、
NH4+,MO2-)
Hydrogen aceptor during respiration without
oxygen (NO3-,SO42-)
Enzyme activator (Cu2+,Mn2+,Zn2+)
Molecular of special structure (Co,Mo)
Physiological function,
? cell composition
? solvent of biochemistry reaction
? chemical and physiological reaction medium
? material transferred medium
? regulate cell temperature
? maintain osmotic pressure
6.Water
existed state:dissociated state(solvent)and integrated
state(structure composition)
◆ based on different carbon sources,
heterotrophs,can’t use CO2 as its mainly or sole carbon energy
autotrophs,can use CO2 as its mainly or sole carbon energy
Section 2 Nutrition types of microbes
◆ based on different energy sources,
phototrophs,obtain energy from light
chemotrophs:obtain energy by oxidation
◆ based on growth factor,
prototroph or wild type
auxotroph
Classification
Nutrition type of microbe
Nutrition type
Energy source
Carbon source
example
photoautotr
ophs
sunlight
CO2
Cyanobacterial，
Purple bacterial,
Green sulfur
bacterial,
Algae
photohetero
trophs
sunlight
CO2 and simple
organic
compounds
Rhodospirillum
chemoautot
rophs
inoganic
compounds
CO2
Nitrobacterial
Sulfbacterial
Ironbacterial
Hydrobacteri
al
chemoheter
otrophs
organic
compounds
organic
compounds
Most
microbes,
Prozotoa
?parasitism —— live in live organisms,
?saprophytism—— live in dead organic,
Chemoheterotrophs
to note that such classification is not absolute,and it may be
changed under different conditions。
? heterotrophs,are dependent on other life forms,can live normally with
at least one kind of organic compound,its carbon soueces and hydrogen
supplier should be in organic form,energy can be obtained by oxidation and
absorption of sunlight,
? autotrophs,can live normally without any organic compounds,
Section 3 Transport mechanisms
for nutrient absorption
The absorbtion of nutrient and excretion of metabolized
products refer to transportion of molecular, nutrient
absorbed in cell and be used,metabolized products secreted
out of the cell,that is the whole process of transportion,
The cell wall can only block large molecular solute
(>600Da) out of the cell,while the membrane which have
lipid bilayer and integral protein is the main
barrier to control the nutrient’s flow,
?permeability is different from absorbtion
?commonly,large molecular first hydrolyze to
small molecular,then be aborbed,
?fat-dissolved,easily permeable
?ionization,the weaker of polar,the faster of the
permeable
I,Simple diffusion
The rate of diffusion is directly proportional
to the concentration gradient of the molecule
across the membrane.；
Do not acquire metabolic energy,is of no
specificity,
Only transformed small molecular,O2,CO2、
glycerol,ethanol and some amino acids；
Lipohilia molecules make use of the
permeability of cell membrane to transport
from high concentration to the low
concentration,
II,Facilitated diffusion
The transported molecular in and out of
the cell have different appetency to the
carrier protein,
characteristic,
Require the presence of specific
carrier protein,down the concentration
gradient
Do not acquire metabolic energy
Transport SO4,PO3,sugars
Carrier protein,
namely permeable enzyme（ mostly are induction-
enzyme）,is specific for one particular molecular or a
group of structurally similar moleculars,It can make the
transportion faster,but is not able to make it against the
concentration gradient,
facilitated diffusion
extracellular
membrane
intracellular
单纯扩散
促进扩散
Concentration gradient
Prapartion of simple diffusion and facilitated diffusion
simple diffusion, facilitated diffusion and active transport：
the transported solute won’t be changed,
III,Active transport
characteristics,
? The main mode for microbes to absorb nutrient
?Against the concentration gradient,require energy
?Need barrier protein,is specific
?Transport organic iron,inorgnic iron,amino acid、
lactose and other sugars
?Need energy to change the conformation of barrier
protein
?Appetence change →protein conformational
change→consume energy
intracellular
Extracellu
lar(outsid
r the
plasma
membrane)
election
transfer
1,Use electron transfer to pump the proton to
extracellular
2,Expel Na+ out of membrane by reverse
transport mechanism
3,Na+ combine with barrier protein complex
4,The form of combination site changed,to
combine with the solute (eg:sugar,amino acid)
5,Barrier protein conformational change,
release Na+ in theintrecellular,then solute
separate from barrier protein,
Mechanism of active transport,use the
gradient of H+ and Na+
characteristics,
? belong to active transport
? solute molecular have chem-modification??
directional phosphorylation
? need a complicated series of transport enzyme
? to transform glucose,fructose,mannan,purin、
nucleotide,fatty acid,et al
IV,Group translocation
The membrane has high impermeability to most phosphate
compounds,
Input each glucose may consume one ATP,
?Mainly depend on PEPand PTS,
PEP+ HPr EI Pyruvic acid+ P-HPr
P-HPr +saccharide EII saccharide-P+HPr
1,Activate the heat stable carrier protein (HPr)
2,Saccharide transferred into membrane after phosphorylation
Transport of glucose group
translocation
2 kinds of systems of PEP and PTS system,
High-energy phosphate transfer from HPr to dissolved EIIA,EIIA combines with EIIB mannan
transport system and separate in the glucose transport system.No matter in which form,phosphate
will first transfer from EIIA to EIIB,and arrive at glycosyl by the process of transport across
membrane,
cytoplast Cytoplasm stroma
I,Definition,
medium,culture medium,Culture medium is the
mixed nutrient suitable for microbe’s growth and breed,
Section 4 culture medium
A,Four principles
1,Definite aims（ different microbes have different nutrient
requirement）,
to culture which microbes,to get which products,useless
II,Principles and methods to select and make culture
medium
2,Suitable nutrients,
pay attention to the concentration and ratio of nutrients，
especially C/N ratio
C/N ratio,mol ratio of carbon atom in carbon sources and
nitrogen atom in nitrogen sources,
C/N ratio in most medium:100/0.5～ 2,
Nitrogen content %,
NH3(82%)>CO(NH2)2(46%)>NH4NO3(35%)>(NH4)2CO3(29.2%)>(NH4)2SO4(21%)
Selection of suitable nutrient
kind example
explaiation
monosaccharides
glucose,maltose,xylose,galactose,
fructose
used as quick carbon sources,but will speed up breathof
microbe,decrease resolved O2,make mid-metabolized products
accumulate,
disaccharide
saccharose,lactose
Used as slow carbon sources,but will speed up breathof
microbe,decrease resolved O2,make mid-metabolized products
accumulate,
polysaccharide
starch,cornmeal,cellulose
slow carbon sources,only few microbes canuse cellulose as
carbon sources
molasses
Sugar beet molasses,sugancane
molasses,
good carbon sources,complicated,usually fermented from yeast
in fermentation industry,widely used in the production of
antibiotic, acetone and butanol.usually include saccharose
35%,glucose 9%,fructose 7%,8kingsother sugars 4%,other
deoxidized materials3%,24 kinds amino acid 4.5%,organic acid
5%,wax,steroids and phosphorous0.4%,ash 12%,water20%,
malt
budded barley
mainly used to culture yeast,can be used to prouduce beer in
industry
grease
cottonseed oil,corn oil,bean
oil,sunflower seed oil
high热值,consume more O2 when cultured in liquid,usually
added as消泡剂 in industry
alcohols
methanol,glycerol
Methanol canbe used to culture yeast
hydrocarbon
CH4,C2H4,C4H10,C12~C20
high热值,consume more O2 when cultured in liquid.hardly
dissolved,don’t too much beating up when cultured in liquid,
organic acid
acetic acid,lactic acid,citric acid
will increase the pH
carbon
sources
stroma
glucose
methane
Normal
paraffin
hydrocar
bon
methanol
ethanol
acetate
fumarat
e
cell
producing
efficiency(
g cell/g
stroma)
0.51
0.62
1.03
0.40
0.68
0.34
0.36
Cell yield by different energy source
Common nitrogen sources for microbe
Kinds
Suitable microbe
Characteristic
organic,beaf extract,yeast extract,
peanut cake power,soybean poder,cotton
cake power,maize slurry,maize protein
power,peptone,yeast power,fish
meal,silkworm pupa power,wheat bran
Most
microbes,eg:Penicillium,ye
ast,E.coli
contain plenty of amino
acid,good nitrogen sources
for microbes
inorganic:ammonium salt,
nitrate,ammonia
Part of microbes(amino
acid heterotrophs aren’t
able to use it)
Nitrate can be used after
deoxidization,may regulate
pH meantime,
N2
few bacteria
(approximately 50 genus,
100 species)
Nitrogen fixation is second
important only to
photosynthesis,
? pH
? osmotic pressure and water ability
? redox potential
3,Suitable chemical and physical conditions,
pH,most suitable pH
v bacteria,pH7.0~8.0
v actinomyces,pH7.0 ~8.5
v yeast,pH3.8~6.0
v mould,pH4.0~6.0
pH value in the medium could change in the growing process
of microorganisms,
v If culture in stroma containing carbohydrate,produce acid,so
the pH will decrease,
v If decompose protein and amino acid,produce NH3,pH will
increase,
v If use (NH4)2SO4 as nitrogen sources,SO4 2- will be
excessive,so the pH decrease,
v If decompose positive iron compounds,eg:NaNO3,Na+ will
be excessive,so the pH increase,
methods to maintain medium pH
stable
Use phosphorus buffer,
K2HPO4 /Na2HPO4,H2PO4/NaH2PO4
Use“standby alkali”, CaCO3,
CaHCO3
Use acidesecent salt,citrate、
lactophosphate et al
Use liquid N2 or HCl,
Osmotic pressure:pressure produced by solutes,when
filterring from high concentration to low cocentration。
Hypertonic solution will cause plasmolysis,
Hypotonic solution will make cell expand or even break,
Utilized ability of water
water activity,aw
aw,the quantity of free or dissociated water that
microbes can utilize under natural environment,
It’s eaqual to the ratio of saturated steam pressure
of the solution and pure water,in the same
temperature and pressure.,
aw =P/Po
P,steam pressure of the solution
Po,steam pressure of pure water
aw of pure water is 1.00,under normal temperature
and pressure
Suitable aw to microbes’ growth is 0.6～ 0.998,
solution
aw
30% glucose
0.964
1% glucose +20%glycerol
0.955
1% glucose +40%saccharose
0.964
Saturated NaCl
0.78
Saturated CaCl2
0.30
Saturated MgCl2
0.30
Saturated LiCl
0.11
Aw of some solution
Eh（ redox potential）,to show the trend of reducer
releasing electron or oxidant accepting eletron in
the redox system,
? Aerobes：＞ + 0.1V。 Generally +0.3~ +0.4V
? Anaerobes,< + 0.1
? Facultative anaerobes,
< +0.1 fermentation
>+0.1 aerobic respiration
4,Appropriate raw material
veconomy principle:use the low grade
replacing high grade,use the waste
replacing normal material,use the
simple replacing complex
veasily treated,with low cost
v produce little waste after treated
Sterilization
Sterilization under high pressure：
1.05kg/cm,1210C,15～ 30min
Sterilization under high temperature
may destroy some nutrients and
change the pH,
B,Four methods
? Ecological simulation
? Lookup literature
? Elaborate
? Experiment Comparison
Category of Culture medium
? Based on the component of the culture medium,
Complex medium ( or Undefined medium )
Complex medium is one kind of culture mediums,which utilizes
animal/ plant/micro-organism or the extract from these
substance,and the components are undefined,e.g,broth
peptone medium,which is a growth medium for bacteria; malt
extract medium,which is a growth medium for yeast,
Advantages,convenience; be rich in nutrition; variety
Defined medium ( or Synthetic medium )
Defined medium is one kind of the culture mediums,which
is compounded by many pure chemical reagent,and the
components ( including the microelement ) are defined
exactly,e.g,Glu-NH4+ medium,which is a growth medium
for bacteria; Starch-NO3- medium,which is a growth
medium for actinomycete,
Expensive, be used in research( metabolism analysis/
genetic analysis)
Advantages, The component is exact; Well-repeat
Semi-defined medium
Solid medium,add solidifying agents; the content of
solidifying agents is 1～ 2％,
Condition of being the solidifying agents,Can’t be utilized or
decomposed by microbe; can keep the solid condition in the
growth temperature range; It isn’t harmful for microbe at
solidification point temperature; can’t be destoried by
sterilization; good-diaphaneity; convenience; cheap,
Solidifying agents that we always use,Agar and Glutin
Usage,isolation/identification/conservation of the strain (Solid
medium allows the surface to form mono-colony )
? Base on the physical condition of culture medium,
Semi-solid medium,the content of solidifying agents is 0.5%
Usage,observe the motion of bacteria; mensurate the titer of
bacteriophage,
Liquid medium, no solidifying agent
Usage,microbe culture ； the research on physiology
/Metabolism
The Difference between Agar and Glutin
Chemistry
Component
Nourish
-ment
Resolv
-ability
Melt
temperature
Solidification
temperature
Concent-
ration
Diaphaneity Adhesio
n value
Tolerance of
Sterilization
under high
pressure
Agar polysaccha-ride
polymer
none infrequent 96℃
40℃
1.5~2
%
high big high
Glutin protein nitrogen
source
easy 25℃ 20℃
5~12
%
high big low
E,Coli growed in EMB (Eosin Methylene Blue) Culture,can
decompose lactose and produce much mixed acid,The thallus
carry with H+,so acidic chromophore Eosin is attracted to
the positively charged molecules of E.coli,Eosin can combine
with Methylene Blue,which makes the colonies purple,Green
metallic luster is observed from the surface glisten of colonies,
Colonies that ferment lactose are red,and colonies that
produce acid weakly are brown,whereas non-fermenters are
transparent,
EMB (Eosin Methylene Blue) Culture medium identified
enteric bacterial
EMB (Eosin Methylene Blue) Culture medium
Brief Summary
1,Microorganisms need about 10 kinds of macroelement,part of
them are used to form carbohydrate, lipid, protein, nucleic
acid,others are bittly used as coenzyme and prosthetic group,
2,All the microorganisms can be classifed on the basis of their
needness of carbon courses, energy sources, hydrogen atom
and electron,
3,Nutrients can’t permeated through cytoplasm membrane
persistently by passive transport,they must choose one kind of
active transport mechanism to do so,
Medium are necessary to culture microbe.You may need to filtrate or
separate special microbes in microorganisms identification, water
quality appraisal, food analysis and so on,remember different aim
determines different medium,
Reviewing question of this chapter
1,What are the macroelement and microelement? 2,How many
kinds of macroelements are needed by microbes?
3,What is nutrition,what is nutrient,What physiological function
does the nutrient have？
4,Try to compare the similarities and differences about nutrients
and nutriention mode among animals, plants and microorganisms,
5,What are carbon sources?List some carbon sources commonly
used by microbes,
6,Find the differences on the using of carbon sources by
microbes,
7,What are nitrogen sources？ List some nitrogen sources
commonly used by microbes,
8,What are the characteristic of the using of nitrogen
sources by microbes？
9,What is amino acid autotrophs,what is amino acid
heterotrophs？
10,Try to compare the differences of the way that
nutrients use to get into cell,
11,What is culture medium and what is complex
medium？
12,What is semi-solid medium？
13,What is differential medium?Give an example of this
kind of medium and explain its principle,
14,What is the C, N ratio？
15,What are the basic principles to make medium?
References,
Zhou Deqin,Microbiological Tutorial,2002,second edition,higher education
press
Kathleen Park Talaro,fundations in microbiology,2005,fifth edition,higher
education press
and Culture medium
Nutrition, Nutrition is a process by which chemical
substances called nutrients are required from the
environment and used for the cellular activities of
metabolism and growth,
Nutrient,the substance could be used by microbes for
metabolism or reproduction of cells,
Nutrient provide the structural substance,energy,
metabolic regulate substance and appropriate
physiological environment,
Some microbes could use nonmaterial energy---light,
I,cellular chemical constitutes
It’s much alike in the life in the element, The main elements
are C,H,O,N (90%---97% to total dry cell weight),C is
about 50%,the others are inorganic nutrient,C/N is 5:1
usually,
Section 1 Six kinds of essential
nutrient for microbes
element
?Macronutrient, C,H,O,N,P,S
?others element,K,Na,Ca,Mg,Fe,Mn、
Cu,Co,Zn,Mo,et al
Existence form,
? organic,protein,sugar,lipoid、
nucleic acid,vitamin,decomposition
substance,metabolic medial products
? inorganic salt ash
? Water---70% ~ 90% to wet cell
weight
element
bacteria
yeast mould
C 50
49.8
47.0
N
15
7.5
5.2
H
8
5.7
6.7
O
20
31.1
40.2
P
3
1.5
1.2
S
1
0.3
0.2
Table 1.The macronutrient content in microbes (%
to dry cell weight)
The substance utilized by generic species
could be utilized by microbes too,
The substance couldn’t be utilized by generic
species could be utilized by microbes,
Some kinds of substance harmful to generic
species also could be utilized by microbes,
Microbes is omnivorous,
II,Main nutrient and the function
Carbon source
Nitrogen source
Energy source
Growth factor
Inorganic salt
Water
Functions,
vconstitute the cells of the microbes
vprovide the energy needed in the
physiological activity to microbes
vthe source to form the microbial
metabolic products
Nutrient is the substance base for
microbial metabolism and living activity,
If lose this base,the life will die,
Heterotrophic microbe,is an organism must obtain its
carbon in an organic form,
Autotrophic microbe,is an organism that uses CO2,an
inorganic gas,as its carbon source,
1.Carbon sources
The nutrient provide the carbon element
for microbies
?Organic carbon sources,protein,nucleic
acid,starch,glucose,et al
?Inorganic acid,CO2,Na2CO3,CaCO3 et al
the carbon sources could be utilized by microbes
(compound)
saccharide,glucose,fructose,maltose,sucrose，
starch,galactose,lactose,mannancellulose，
hemicellulose,lignin,et al
organic acid,lactic acid,citric acid，,short chain
fatty acid,long chain fatty acid, amino acid,et al
alcoholic aldehyde,ethanol,et al
fat,fat,lecithoid,et al
hydrocarbon,natural gas,petroleum,olefin,et al
CO2
carbonate,NaHCO3,CaCO3,chalk,et al
others,cyanide,protein,peptide,nucleic acid
?organic nitrogen sources, peptone,soybean powder
,maize slurry
?inorganic nitrogen sources,NH4NO3,(NH4)2SO4
?gas nitrogen sources,N
2 in the atmosphere
2.Nitrogen sources
The nutrient provide the nitrogen element for microbes,
Normally,nitrogen source couldn’t be used as energy sources,
?quick nitrogen sources
?slow nitrogen sources
energy sources are the substances or radiant energy that could provide
primal energy sources, Heterotrophs’s carbon sources could also be its
energy sources,
3,Energy sources
Inorganic:energy to chemoautotrophs
Energy sources
table
Chemical substances
Radiant energy,energy to photoautotrophs and
photoheterotrophs,
Organic,energy to chemoheterotrophs
mono-function, radiant energy
?di-function,reductive inorganic nutrient,such as NH4+ is the
energy sources as well as the nitrogen sources of nitrate
bacterial,
?triple function,the nutrients including N, C, H, O are
energy sources, carbon sources and nitrogen sources
heterotrophs microbes,
one kind of nutrient could provide more than one kind of function
Definition:An organic compound such as an amino
acid,nitrogen base,or vitamin that cannot be
synthesized by an organism and must be provided as
a nutrient is a growth factor,
Sources of growth factors in culture medium,
yeast extract,maize slurry,wort,et al。
4,Growth factor
Function,coenzyme or necessary to activation of
enzyme,
narrow sense,vitamin
broad sense,vitamin,amino acid,base,fatty
acid,et al
?auxoautotrophs
?auxoheterotrophs
?nutritional deficiency mutant
?auxo-excessivetrophs
vitamin
species
thiamin B
1
Bacillus anthracis
lactoflavin
Clostridium tetani
nicotinic acid
Brucella abortus
vitamin B6
Lactobacillus spp,
biotin
Leuconostoc mesenteroides
pantothenic acid
Proteus morganii
folacin
Leuconostoc dextranicum
B12
Lactobacillus spp,
vitamin K
Bacteroides melaninogenicus
Vitamin necessary to some
bacterials
5,Inorganic salts
Macroelement,need the concentration of
the element at 10-3-10-4M
Microelement,need the concentration of
the element at 10-6-10-8M
The physiology function for inorganic salts
inorganic
salts
Macroelement
Microelement
Usual
function
Especial
function
Normal molecular in cell(P,S,Ca,Mg,Fe)
Physiology
modulation material
maintain osmotic pressure (Na+)
enzyme activator(Mg2+)
stablize pH
Energy sourse of chemoautotrophic bacteria (S,Fe2+、
NH4+,MO2-)
Hydrogen aceptor during respiration without
oxygen (NO3-,SO42-)
Enzyme activator (Cu2+,Mn2+,Zn2+)
Molecular of special structure (Co,Mo)
Physiological function,
? cell composition
? solvent of biochemistry reaction
? chemical and physiological reaction medium
? material transferred medium
? regulate cell temperature
? maintain osmotic pressure
6.Water
existed state:dissociated state(solvent)and integrated
state(structure composition)
◆ based on different carbon sources,
heterotrophs,can’t use CO2 as its mainly or sole carbon energy
autotrophs,can use CO2 as its mainly or sole carbon energy
Section 2 Nutrition types of microbes
◆ based on different energy sources,
phototrophs,obtain energy from light
chemotrophs:obtain energy by oxidation
◆ based on growth factor,
prototroph or wild type
auxotroph
Classification
Nutrition type of microbe
Nutrition type
Energy source
Carbon source
example
photoautotr
ophs
sunlight
CO2
Cyanobacterial，
Purple bacterial,
Green sulfur
bacterial,
Algae
photohetero
trophs
sunlight
CO2 and simple
organic
compounds
Rhodospirillum
chemoautot
rophs
inoganic
compounds
CO2
Nitrobacterial
Sulfbacterial
Ironbacterial
Hydrobacteri
al
chemoheter
otrophs
organic
compounds
organic
compounds
Most
microbes,
Prozotoa
?parasitism —— live in live organisms,
?saprophytism—— live in dead organic,
Chemoheterotrophs
to note that such classification is not absolute,and it may be
changed under different conditions。
? heterotrophs,are dependent on other life forms,can live normally with
at least one kind of organic compound,its carbon soueces and hydrogen
supplier should be in organic form,energy can be obtained by oxidation and
absorption of sunlight,
? autotrophs,can live normally without any organic compounds,
Section 3 Transport mechanisms
for nutrient absorption
The absorbtion of nutrient and excretion of metabolized
products refer to transportion of molecular, nutrient
absorbed in cell and be used,metabolized products secreted
out of the cell,that is the whole process of transportion,
The cell wall can only block large molecular solute
(>600Da) out of the cell,while the membrane which have
lipid bilayer and integral protein is the main
barrier to control the nutrient’s flow,
?permeability is different from absorbtion
?commonly,large molecular first hydrolyze to
small molecular,then be aborbed,
?fat-dissolved,easily permeable
?ionization,the weaker of polar,the faster of the
permeable
I,Simple diffusion
The rate of diffusion is directly proportional
to the concentration gradient of the molecule
across the membrane.；
Do not acquire metabolic energy,is of no
specificity,
Only transformed small molecular,O2,CO2、
glycerol,ethanol and some amino acids；
Lipohilia molecules make use of the
permeability of cell membrane to transport
from high concentration to the low
concentration,
II,Facilitated diffusion
The transported molecular in and out of
the cell have different appetency to the
carrier protein,
characteristic,
Require the presence of specific
carrier protein,down the concentration
gradient
Do not acquire metabolic energy
Transport SO4,PO3,sugars
Carrier protein,
namely permeable enzyme（ mostly are induction-
enzyme）,is specific for one particular molecular or a
group of structurally similar moleculars,It can make the
transportion faster,but is not able to make it against the
concentration gradient,
facilitated diffusion
extracellular
membrane
intracellular
单纯扩散
促进扩散
Concentration gradient
Prapartion of simple diffusion and facilitated diffusion
simple diffusion, facilitated diffusion and active transport：
the transported solute won’t be changed,
III,Active transport
characteristics,
? The main mode for microbes to absorb nutrient
?Against the concentration gradient,require energy
?Need barrier protein,is specific
?Transport organic iron,inorgnic iron,amino acid、
lactose and other sugars
?Need energy to change the conformation of barrier
protein
?Appetence change →protein conformational
change→consume energy
intracellular
Extracellu
lar(outsid
r the
plasma
membrane)
election
transfer
1,Use electron transfer to pump the proton to
extracellular
2,Expel Na+ out of membrane by reverse
transport mechanism
3,Na+ combine with barrier protein complex
4,The form of combination site changed,to
combine with the solute (eg:sugar,amino acid)
5,Barrier protein conformational change,
release Na+ in theintrecellular,then solute
separate from barrier protein,
Mechanism of active transport,use the
gradient of H+ and Na+
characteristics,
? belong to active transport
? solute molecular have chem-modification??
directional phosphorylation
? need a complicated series of transport enzyme
? to transform glucose,fructose,mannan,purin、
nucleotide,fatty acid,et al
IV,Group translocation
The membrane has high impermeability to most phosphate
compounds,
Input each glucose may consume one ATP,
?Mainly depend on PEPand PTS,
PEP+ HPr EI Pyruvic acid+ P-HPr
P-HPr +saccharide EII saccharide-P+HPr
1,Activate the heat stable carrier protein (HPr)
2,Saccharide transferred into membrane after phosphorylation
Transport of glucose group
translocation
2 kinds of systems of PEP and PTS system,
High-energy phosphate transfer from HPr to dissolved EIIA,EIIA combines with EIIB mannan
transport system and separate in the glucose transport system.No matter in which form,phosphate
will first transfer from EIIA to EIIB,and arrive at glycosyl by the process of transport across
membrane,
cytoplast Cytoplasm stroma
I,Definition,
medium,culture medium,Culture medium is the
mixed nutrient suitable for microbe’s growth and breed,
Section 4 culture medium
A,Four principles
1,Definite aims（ different microbes have different nutrient
requirement）,
to culture which microbes,to get which products,useless
II,Principles and methods to select and make culture
medium
2,Suitable nutrients,
pay attention to the concentration and ratio of nutrients，
especially C/N ratio
C/N ratio,mol ratio of carbon atom in carbon sources and
nitrogen atom in nitrogen sources,
C/N ratio in most medium:100/0.5～ 2,
Nitrogen content %,
NH3(82%)>CO(NH2)2(46%)>NH4NO3(35%)>(NH4)2CO3(29.2%)>(NH4)2SO4(21%)
Selection of suitable nutrient
kind example
explaiation
monosaccharides
glucose,maltose,xylose,galactose,
fructose
used as quick carbon sources,but will speed up breathof
microbe,decrease resolved O2,make mid-metabolized products
accumulate,
disaccharide
saccharose,lactose
Used as slow carbon sources,but will speed up breathof
microbe,decrease resolved O2,make mid-metabolized products
accumulate,
polysaccharide
starch,cornmeal,cellulose
slow carbon sources,only few microbes canuse cellulose as
carbon sources
molasses
Sugar beet molasses,sugancane
molasses,
good carbon sources,complicated,usually fermented from yeast
in fermentation industry,widely used in the production of
antibiotic, acetone and butanol.usually include saccharose
35%,glucose 9%,fructose 7%,8kingsother sugars 4%,other
deoxidized materials3%,24 kinds amino acid 4.5%,organic acid
5%,wax,steroids and phosphorous0.4%,ash 12%,water20%,
malt
budded barley
mainly used to culture yeast,can be used to prouduce beer in
industry
grease
cottonseed oil,corn oil,bean
oil,sunflower seed oil
high热值,consume more O2 when cultured in liquid,usually
added as消泡剂 in industry
alcohols
methanol,glycerol
Methanol canbe used to culture yeast
hydrocarbon
CH4,C2H4,C4H10,C12~C20
high热值,consume more O2 when cultured in liquid.hardly
dissolved,don’t too much beating up when cultured in liquid,
organic acid
acetic acid,lactic acid,citric acid
will increase the pH
carbon
sources
stroma
glucose
methane
Normal
paraffin
hydrocar
bon
methanol
ethanol
acetate
fumarat
e
cell
producing
efficiency(
g cell/g
stroma)
0.51
0.62
1.03
0.40
0.68
0.34
0.36
Cell yield by different energy source
Common nitrogen sources for microbe
Kinds
Suitable microbe
Characteristic
organic,beaf extract,yeast extract,
peanut cake power,soybean poder,cotton
cake power,maize slurry,maize protein
power,peptone,yeast power,fish
meal,silkworm pupa power,wheat bran
Most
microbes,eg:Penicillium,ye
ast,E.coli
contain plenty of amino
acid,good nitrogen sources
for microbes
inorganic:ammonium salt,
nitrate,ammonia
Part of microbes(amino
acid heterotrophs aren’t
able to use it)
Nitrate can be used after
deoxidization,may regulate
pH meantime,
N2
few bacteria
(approximately 50 genus,
100 species)
Nitrogen fixation is second
important only to
photosynthesis,
? pH
? osmotic pressure and water ability
? redox potential
3,Suitable chemical and physical conditions,
pH,most suitable pH
v bacteria,pH7.0~8.0
v actinomyces,pH7.0 ~8.5
v yeast,pH3.8~6.0
v mould,pH4.0~6.0
pH value in the medium could change in the growing process
of microorganisms,
v If culture in stroma containing carbohydrate,produce acid,so
the pH will decrease,
v If decompose protein and amino acid,produce NH3,pH will
increase,
v If use (NH4)2SO4 as nitrogen sources,SO4 2- will be
excessive,so the pH decrease,
v If decompose positive iron compounds,eg:NaNO3,Na+ will
be excessive,so the pH increase,
methods to maintain medium pH
stable
Use phosphorus buffer,
K2HPO4 /Na2HPO4,H2PO4/NaH2PO4
Use“standby alkali”, CaCO3,
CaHCO3
Use acidesecent salt,citrate、
lactophosphate et al
Use liquid N2 or HCl,
Osmotic pressure:pressure produced by solutes,when
filterring from high concentration to low cocentration。
Hypertonic solution will cause plasmolysis,
Hypotonic solution will make cell expand or even break,
Utilized ability of water
water activity,aw
aw,the quantity of free or dissociated water that
microbes can utilize under natural environment,
It’s eaqual to the ratio of saturated steam pressure
of the solution and pure water,in the same
temperature and pressure.,
aw =P/Po
P,steam pressure of the solution
Po,steam pressure of pure water
aw of pure water is 1.00,under normal temperature
and pressure
Suitable aw to microbes’ growth is 0.6～ 0.998,
solution
aw
30% glucose
0.964
1% glucose +20%glycerol
0.955
1% glucose +40%saccharose
0.964
Saturated NaCl
0.78
Saturated CaCl2
0.30
Saturated MgCl2
0.30
Saturated LiCl
0.11
Aw of some solution
Eh（ redox potential）,to show the trend of reducer
releasing electron or oxidant accepting eletron in
the redox system,
? Aerobes：＞ + 0.1V。 Generally +0.3~ +0.4V
? Anaerobes,< + 0.1
? Facultative anaerobes,
< +0.1 fermentation
>+0.1 aerobic respiration
4,Appropriate raw material
veconomy principle:use the low grade
replacing high grade,use the waste
replacing normal material,use the
simple replacing complex
veasily treated,with low cost
v produce little waste after treated
Sterilization
Sterilization under high pressure：
1.05kg/cm,1210C,15～ 30min
Sterilization under high temperature
may destroy some nutrients and
change the pH,
B,Four methods
? Ecological simulation
? Lookup literature
? Elaborate
? Experiment Comparison
Category of Culture medium
? Based on the component of the culture medium,
Complex medium ( or Undefined medium )
Complex medium is one kind of culture mediums,which utilizes
animal/ plant/micro-organism or the extract from these
substance,and the components are undefined,e.g,broth
peptone medium,which is a growth medium for bacteria; malt
extract medium,which is a growth medium for yeast,
Advantages,convenience; be rich in nutrition; variety
Defined medium ( or Synthetic medium )
Defined medium is one kind of the culture mediums,which
is compounded by many pure chemical reagent,and the
components ( including the microelement ) are defined
exactly,e.g,Glu-NH4+ medium,which is a growth medium
for bacteria; Starch-NO3- medium,which is a growth
medium for actinomycete,
Expensive, be used in research( metabolism analysis/
genetic analysis)
Advantages, The component is exact; Well-repeat
Semi-defined medium
Solid medium,add solidifying agents; the content of
solidifying agents is 1～ 2％,
Condition of being the solidifying agents,Can’t be utilized or
decomposed by microbe; can keep the solid condition in the
growth temperature range; It isn’t harmful for microbe at
solidification point temperature; can’t be destoried by
sterilization; good-diaphaneity; convenience; cheap,
Solidifying agents that we always use,Agar and Glutin
Usage,isolation/identification/conservation of the strain (Solid
medium allows the surface to form mono-colony )
? Base on the physical condition of culture medium,
Semi-solid medium,the content of solidifying agents is 0.5%
Usage,observe the motion of bacteria; mensurate the titer of
bacteriophage,
Liquid medium, no solidifying agent
Usage,microbe culture ； the research on physiology
/Metabolism
The Difference between Agar and Glutin
Chemistry
Component
Nourish
-ment
Resolv
-ability
Melt
temperature
Solidification
temperature
Concent-
ration
Diaphaneity Adhesio
n value
Tolerance of
Sterilization
under high
pressure
Agar polysaccha-ride
polymer
none infrequent 96℃
40℃
1.5~2
%
high big high
Glutin protein nitrogen
source
easy 25℃ 20℃
5~12
%
high big low
E,Coli growed in EMB (Eosin Methylene Blue) Culture,can
decompose lactose and produce much mixed acid,The thallus
carry with H+,so acidic chromophore Eosin is attracted to
the positively charged molecules of E.coli,Eosin can combine
with Methylene Blue,which makes the colonies purple,Green
metallic luster is observed from the surface glisten of colonies,
Colonies that ferment lactose are red,and colonies that
produce acid weakly are brown,whereas non-fermenters are
transparent,
EMB (Eosin Methylene Blue) Culture medium identified
enteric bacterial
EMB (Eosin Methylene Blue) Culture medium
Brief Summary
1,Microorganisms need about 10 kinds of macroelement,part of
them are used to form carbohydrate, lipid, protein, nucleic
acid,others are bittly used as coenzyme and prosthetic group,
2,All the microorganisms can be classifed on the basis of their
needness of carbon courses, energy sources, hydrogen atom
and electron,
3,Nutrients can’t permeated through cytoplasm membrane
persistently by passive transport,they must choose one kind of
active transport mechanism to do so,
Medium are necessary to culture microbe.You may need to filtrate or
separate special microbes in microorganisms identification, water
quality appraisal, food analysis and so on,remember different aim
determines different medium,
Reviewing question of this chapter
1,What are the macroelement and microelement? 2,How many
kinds of macroelements are needed by microbes?
3,What is nutrition,what is nutrient,What physiological function
does the nutrient have？
4,Try to compare the similarities and differences about nutrients
and nutriention mode among animals, plants and microorganisms,
5,What are carbon sources?List some carbon sources commonly
used by microbes,
6,Find the differences on the using of carbon sources by
microbes,
7,What are nitrogen sources？ List some nitrogen sources
commonly used by microbes,
8,What are the characteristic of the using of nitrogen
sources by microbes？
9,What is amino acid autotrophs,what is amino acid
heterotrophs？
10,Try to compare the differences of the way that
nutrients use to get into cell,
11,What is culture medium and what is complex
medium？
12,What is semi-solid medium？
13,What is differential medium?Give an example of this
kind of medium and explain its principle,
14,What is the C, N ratio？
15,What are the basic principles to make medium?
References,
Zhou Deqin,Microbiological Tutorial,2002,second edition,higher education
press
Kathleen Park Talaro,fundations in microbiology,2005,fifth edition,higher
education press
