Download Selective and Differential Media (I) 选择和鉴别培养基

Chapter 4: Nutrition and Media
第四章：微生物营养和陪养基
The whole of nature, as has been said, is a conjugation of the verb to eat, in the
active and passive.
- William Ralph Inge
Content
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Nutrient requirement for microorganism
Transferring of substance into cells
Different medium for specific purpose
Nutrition营养
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Nutrition 营养(作用) include:
– Substance 物质
– Energy 能量
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Nutrient 营养(物):
Substance of nutritional values
Nutritional requirements at elementary level在元素水平上的营养需求
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Revealed by cell's elemental composition
– Macro-: C, H, O, N, S. P, K, Mg, Fe, Ca
– Micro-: Mn, Zn, Ni, Co钴, Cu, Mo钼
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Forms: Water, inorganic ions无机离子, small molecules, and macromolecules
Either a structural or functional role
Major elements, their sources and functions in bacterial cells
Nutrient Requirements of Microbes微生物的六种营养要素
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Carbon source 碳源
Nitrogen source 氮源
Energy 能量
Growth factor 生长因子
Inorganic substance 无机物
Water 水
1. Carbon Sources 碳源
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Carbon requirements:
– A wide range, may also be energy source
– Organic carbon, CO2 or CO, methane甲烷
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Heterotrophs 异养生物: organic carbon
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Autotrophs 自养生物: CO2 as primary or sole carbon source
Nutrient Requirements of Microbes
Carbohydrate Catabolism
碳水化合物分解代谢
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Carbohydrates oxidized as their primary source of energy 糖类作为主要能源
Glucose: most common energy source葡萄糖是最普遍的能源
– Respiration 呼吸作用
– Fermentation 发酵作用
2. ENERGY 能量
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Light (the sun or lamps)
Inorganic substances
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Sulfur 硫, CO or ammonia 氨
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Preformed organic matter
Sugar, protein, fats etc.
Energy Sources for Bacterial Growth
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All living organisms require energy source
– Phototrophs光能自养菌: radiant energy (light)
– Heterotrophs 异养生物 = chemo(hetero)trophs 化能异养: use (oxidize)
organic carbon form
– Lithotrophs 无机营养生物= chemoautotrophs 化能自养: oxidize inorganic
compounds
4 major nutritional types of procaryotes on the basis of carbon and energy
sources
原核生物的四种营养类型
4 major nutritional types of procaryotes on the basis of carbon and energy
sources
原核生物的四种营养类型
CRITICAL THINKING QUESTION
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How would you go about isolating a microbe that used gasoline 汽油 for a
food?
Exxon Valdez Oil Spill
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Approximately 11 million gallons of oil were spilled into the waters of Prince
William Sound during the 1989 Exxon Valdez Oil Spill Prince
– William Sound Oil Spill Recovery Institute
MEOR
Microbial enhanced oil recovery (MEOR)
• In situ generation of CO2 for pressure and solubilization增溶
• microbial production of organic acids
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conversion of hydrocarbons to lower MW
production of surface active agents
encouragement of consolidation凝固 of oil to droplets
enzymatic modification of hydrocarbons
viscosity improvement
3. NITROGEN Source 氮源
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N2, ammonia 氨, nitrate/nitrite 硝酸盐, or a nitrogenous 含氮的 organic
compound (protein or nucleic acid)
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Classification based on AA requirement
– Amino-acid-autotrophs氨基酸自养生物:SCP
– Amino-acid-heterotrophs 氨基酸异养生物
4. Inorganic Compounds 无机物
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Large amount: 10-3~10-4mol/L
– P. S. K, Mg, Ca, Na, Fe
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TRACE metals 微量金属: 10-6~10-8mol/L,
– required by some enzymes
– Include: Cu, Zn 锌, Co钴, Mn, Mo
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E.g. K2HPO4, MgSO4:
– supply 4 elements of greatest amount
5. Growth factors 生长因子
• Definition: Small amounts of certain essential organic compounds unable to be
synthesized
• Required to fulfill specific roles in metabolism
• Results from either a blocked or missing metabolic pathway 起因于阻断或丢
失的代谢途径
– Auxoautotrophs 生长因子自养型微生物
– e.g E. coli
– Auxoheterotrophs 生长因子异养型微生物:
– e.g. Lactobacillus乳酸杆菌
Categories of Growth Factors
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Purines and pyrimidines 嘌呤与嘧啶:
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synthesis of nucleic acids (DNA and RNA)
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Amino acids 氨基酸:
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synthesis of proteins
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Vitamins 维生素:
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as coenzymes and functional groups of certain enzymes
Question?
Why are amino acid, purines, and pyrimidines often growth factors, whereas
glucose is usually not?
Nutritional types of major organisms
主要生物体的营养类型
• Eukaryotes 真核生物:
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Photoautotrophic 光能自养(e.g. plants & algae)
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heterotrophic 异养 (e.g. animals, protozoa原生动物, fungi)
• Lithotrophy无机自养:
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unique to procaryotes
• Photoheterotrophy光能异养:
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common in purple & green bacteria, only a few eukaryotic algae
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Phototrophy 光能营养: not found in Archaea
Different nutritional requirements
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Vastly different nutritional requirements
– E. coli: capable of synthesizing all organic compounds it needs
– Leuconostoc mesenteroides 肠膜明串珠菌: need many externally provided
organic compounds - extremely fastidious (nutritionally demanding)
Fastidious orgnisms 需要复杂营养的生物体
• Require PREFORMED organic molecules like vitamins, AA, nucleic acids, carbohydrates;
• In general pathogens need more PREFORMED organic molecules than non-pathogens
• A simple rule of thumb:
– "if humans can use something for food, many microbes will also love it". - humans are
fastidious.
– The reverse is not always true
CRITICAL THINKING QUESTION
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Why does it make sense from a nutritional standpoint 营养观点 that most
pathogens are nutritionally fastidious?
Nutrient Transportation
营养物质进入细胞方式
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Cytoplasmic membrane: selective transportation
– Proteins in the membrane:
• typically span the whole membrane, contacting with the outside
environment and cytoplasm.
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often require the expenditure of energy
Four basic types of transport systems 四种传输方式
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Simple (passive) Diffusion 单纯 (被动) 运输
Facilitated Diffusion 促进扩散
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Group Translocation 基团转移
Active Transport 主动运输
Passive diffusion 单纯 (被动)扩散
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Directed by laws of simple diffusion.
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Involves a protein to transport – specific
No transport protein involved
Nonspecific, energy is not required
A concentration gradient not be generated.
Figure 8.9 The diffusion of solutes across membranes
Figure 8.11 The water balance of living cells shriveled shrink lysed
Facilitated Diffusion 促进扩散
Solutes: not concentrated against gradient
Energy: not required
Not widely used in prokaryotes
Figure 8.13 One model for facilitated diffusion
Passive & Facilitated Diffusion
Membrane-spanning transporters
穿膜运输蛋白
• In prokaryotes, membrane-spanning transporters typically contain 12 alpha helices that
align with each other in a circle to form a channel through the membrane. 原核生物
的穿膜运输蛋白由12个螺旋形成穿膜的通道
Three individual transporters
Group translocation 基团转位
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Protein-substrate: chemical modification
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Target NOT altered & accumulation of target occurs
– inside conc. > external conc.
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Proteins: molecular pumps against a concentration gradient
No actual concentration takes place
Most requires energy.
Catabolic pathways sometime use
– Efficient: bring substrate into the cell and begin the breakdown process
PEP  glucose
Mechanism of the phosphotransferase system
Active transport 主动运输
Energy source: ATP or proton motive force (PMF)
Figure An electrogenic pump
Figure Cotransport
Simple transporter: Lac permease
Figure The sodium-potassium pump: a specific case of active transport
Removal of phospho-head groups of membrane lipids immobilizes voltage sensors of
K+ channels
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Nature 451, 826-829 (14 February 2008)
Figure Review: passive and active transport compared
Summary of bacterial transport systems
Discussion?
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Why do microorganisms normally take up nutrients using transport proteins?
What advantage is active transport compared to facilitated diffusion?
5 min each
Manner to cultivate bacteria 细菌培养方式
• Liquid media 液体培养基:
– used for growth of pure batch cultures 纯批量培养物生长: fermentation
industry 发酵工业
• Solidified media 固化培养基:1~2% agar
– Isolation of pure cultures 纯培养物分离
– Estimate viable bacterial populations 测活菌数
• Semisolid medium半固体培养基: 0.5% agar
Culture Medium
• Function:
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Isolation & maintenance of pure cultures 纯培养的分离和保存
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Identification 鉴定细菌
• Example
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Basal medium 基底培养基
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Minimal medium
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NNN medium with blood含琼脂、盐及兔血的培养基for trypanosoma锥虫属
Principles of medium design and selection 培养基设计与选择的原则
• Objective 目的明确
– Bacterial mass or metabolism products?
• Balanced nutrition 营养协调
– Ten-time-rule:
H2O > C+energy > N source > P, S > K,Mg > growth factor
• Appropriate physical & chemical condition
– pH:
• Bacteria: 7.0 ~ 8.0; Acetenomycets: 7.5 ~ 8.5
• Yeast: 3.8 ~ 6.0; Mould: 4.0 ~ 5.8
Osmotic pressure & aw: optimal aw 0.6~0.998
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• Economic 经济节约
Agar琼脂 - the usual gelling agent胶凝剂
• For solid or semisolid medium
• A hydrocolloid水状胶体derived from red algae
• Unique physical properties:
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melts at 100 degrees
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remains liquid until cooled to 45C at which it gels
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Not metabolized by most bacteria – relatively inert
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simply holds (gels) nutrients in aquaeous solution.
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Nontoxic to most organisms
Categories of Culture medium based on the composition 培养基分类
• Chemically-defined(synthetic)medium组合培养基
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Exact chemical composition is known
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For growing fastidious organisms & nutritional requirements
• Complex (undefined) medium天然复合培养基
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Composition is not known
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contain biological origin (blood, milk, yeast or beef extract) - full range of
growth factors
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For most bacteria and fungi
• Semi-defined medium 半组合培养基
A chemically-defined medium for growth of heterotrophic bacterium (pH 7.0) 异养菌
Defined enrichment medium富集培养基 for Thiobacillus硫杆菌属thiooxidans, a
lithoautotrophic bacterium (pH 3.0) 无机自养菌
Complex medium for the growth of fastidious bacteria (pH 6.6)
Medium for some specific bacteria
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Pathogenic bacteria of animals:
– complex medium复合培养基
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A few fastidious pathogens
– Treponema pallidum 梅毒螺旋体, Mycobacterium leprae 麻风分支杆菌:
artificial culture media & conditions not been established.
Selective Media 选择型培养基
• Inhibits some bacteria while selecting others
• Example: Brilliant Green Agar 碱性亮绿琼脂
– Dyes: inhibit the growth of Gram (+)
– Selects for G (-) bacteria: most G.I. Tract infections
Examples of Selective Media
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EMB (Eosin Methylene Blue 伊红美兰培养基)
– Dyes inhibit Gram (+) bacteria
– Selects Gram (-) bacteria, especially E. coli
– G.I. Tract infections
caused by Gram (-)
深紫色，绿色金属闪光
Differential Media 鉴别培养基
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Differentiates different organisms growing on the same plate
Example: Blood Agar Plates (TSA with 5% sheep blood) 血琼脂平板
– differentiate different types of Streptococci 链球菌
Differential Media 鉴别培养基 for Hemolytic Streptococci 溶血链球菌
• Blood agar plates (TSA 大豆胰蛋白胨琼脂 with 5% sheep blood) 血琼脂平板
• lysis of RBC’s or not
– Alpha: Incomplete lysis
– Beta: Complete lysis
– Gamma: No lysis
Selective and Differential Media (I) 选择和鉴别培养基
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Mannitol Salt Agar 甘露醇盐琼脂
– Identify Staphylococcus aureus 金黄色葡萄球菌
– High salt conc. (7.5%) inhibits most bacteria
– Sugar: Mannitol
– pH indicator (→yellow when acid)
Selective & Differential Media (II)
• MacConkey’s Agar
– used to identify Salmonella 沙门菌: pH Indicator
– Bile salts 胆盐 & crystal violet: inhibits G+
– Lactose 乳糖
• Many Gram (-) enteric小肠的 non-pathogenic bacteria can ferment
lactose, Salmonella can not
Differentiation of C. diphtheriae 白喉杆菌
Selective enrichment medium for extreme halophiles 耐盐性生物富集培养基 (pH
7.4)
TYPES OF CULTURE MEDIA
Critical Thinking
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You have two microorganisms that grow well in nutrient agar. Does that mean
they would both grow well in the same minimal medium? Why?
Key Points
• Six major types of nutrients for microorganism 六大营养要素
• Four major nutritional types of procaryotes on the basis of carbon and energy
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sources
Growth factor and its major categories
Four basic types of nutrient transport systems
Difference between synthetic medium and complex medium 合成与天然培养
基的区别
Application of selective medium and differential medium 选择与鉴别培养基的
应用