Topic 2 Microbial metabolism (Ch. 4) Nutrition and growth (Ch.

5)

Microbial metabolism

Metabolism = sum of all chemical reactions in a living organism All chemical reactions release or require energy Metabolism = energy balancing act

Microbial metabolism
Catabolism: release energy, breakdown of complex organic compounds simpler ones, hydrolytic (use H2O, chemical bonds broken), exergonic (net energy production) Anabolism: require energy, building of complex organic molecules from simpler ones, dehydration reactions (release H2O), endergonic (net energy consumption), biosynthetic

Microbial metabolism

Interrelationship of metabolic pathways

Cell wall Cytoplasmic membrane
Polysaccharides Peptidoglycan

Phospholipids Glucose G-3-P Pyruvate

Amino acids

Ribose-P

Nucleotides

DNA

Fatty Acids

Acetyl-CoA
Amino acids

RNA

ENERGY

CAC

Proteins

Microbial metabolism

Energy production
Anabolic reactions: ATP ADP + Pi + energy Catabolic reactions: ADP + Pi + energy ATP

Energy production
Energy stored in the chemical bonds of ATP High energy bonds = unstable bonds Energy released quickly and easily Used for anabolic reactions

Energy production
How much energy is needed by E. coli?
Cell constituent Number of molecules per cell Molecules synthesised per second Molecules of ATP required per second for synthesis

DNA RNA Polysaccharides Lipids Proteins

1 15,000 39,000 15,000,000 1,700,000

0.00037 12.5 32.5 12,500 1,400

60,000 75,000 65,000 87,000 2,120,000

Synthesis of a single DNA molecule (i.e. one replication) will consume approximately 162,000,000 ATP molecules

Energy production
Energy produced in two general ways Oxidation-reduction ATP generation Oxidation = removal of electrons from atom or molecule, often produces energy Reduction is the gain of one or more electrons Oxidation and reduction are coupled reactions, one molecule is oxidised, another is reduced = redox reactions

Energy production

Cellular oxidation involves simultaneous loss of electron and proton (H+ and e-), electrons cannot exist free in solution; loss of H atoms = hydrogenation

Energy production
Co-enzymes help in the oxidation of organic substrates by accepting protons (other co-enzymes donate electrons) Nicotinamide adenine dinucleotide (NAD+) Nicotinamide adenine dinucleotide phosphate (NADP+) = important cellular co-enzymes (derived from vitamins, B vitamins) NAD+ primarily involved in catabolic reactions NADP+ primarily involved in anabolic reactions

Energy production
Organic molecule + NAD+
Donates two H atoms (2 X [H+ + e-])

Oxidised organic molecule + NADH + H+

NAH+ receives one H atom and one eNADH contains more energy, used to generate ATP in later reactions

Energy production
Biological oxidation-reduction degrades highly reduced compounds (nutrient molecules with many H atoms) to highly oxidised compounds Glucose CO2 + H2O + energy (converted to ATP) [ADP + Pi + energy ATP] Addition of P = phosphorylation Three mechanisms of phosphorylation in organisms

Energy production
Substrate-level phosphorylation: High-energy Pi directly transferred from phosphorylated compound to ADP Sub-Pi + ADP Sub + ATP Oxidative phosphorylation: Electrons transferred from organic compounds to electron carriers (NAD+, FAD) Electrons passed through series of different carriers to O2 or other inorganic molecules (electron transport system) in plasma membrane (prokaryotes) or inner mitochondrial membrane (eukaryotes) Electron transfer releases energy, generates ATP

Energy production
Photophosphorylation: Occurs in photosynthetic cells, light-trapping pigments (chlorophyll) Organic molecules (sugars) synthesised with energy from light using CO2 and H2O Light energy converted to chemical energy (ATP and NADPH), used to synthesise organic molecules Involves electron transport system

Carbohydrate catabolism
Carbohydrates are primary source of energy in most microorganisms Breakdown of carbohydrate molecules to produce energy is of great importance in cell metabolism Glucose is the most common carbohydrate energy source Microorganisms can also catabolise lipids and proteins energy

Carbohydrate catabolism
Microorganisms derive energy from carbohydrates by three processes: 1. Aerobic respiration 2. Anaerobic respiration 3. Fermentation (anaerobic)
1

Glucose Pyruvate
Energy +++ ++ + 3

Citric acid cycle

2

Electron Transport (O2) Electron Transport (Not O2)

(Terminal electron acceptors)

[Citric acid Cycle] (Organic compounds)

1= 2= 3=

Carbohydrate catabolism
Both respiration and fermentation begin with glycolysis (oxidation of glucose to pyruvate, some ATP and NADH produced)

Respiration: Citric acid cycle (CAC) - oxidation of acetyl CoA (derivative of pyruvate) to CO2, some ATP, NADH and FADH2 produced) Electron transport system - NADH and FADH2 oxidised, cascade of redox reactions energy, generates considerable amount of ATP

Carbohydrate catabolism
Fermentation: No CAC or electron transport, much lower ATP yield Pyruvate converted to different products, depending on microorganism (e.g. alcohol, lactic acid, other acids) Occurs in absence of oxygen (anaerobic)

Glycolysis
Also called the Emden-Myerhoff pathway Enzymes catalyse the splitting of glucose (6-carbon sugar) into two 3-carbon sugars The 3-carbon sugars are oxidised 2X pyruvate NAD+ reduced to NADH, net production of two ATP molecules (substrate level phosphorylation) Can occur in presence or absence of oxygen Two stages: preparatory reactions and oxidation (energy-conserving) reactions

Glycolysis
Preparatory reactions
Glucose

ATP ADP

-P

Glucose-6-phosphate

-P

Fructose-6-phosphate

Glycolysis
Preparatory reactions -P
Fructose-6-phosphate

ATP ADP P-P

Fructose-1,6-phosphate

Dihydroxyacetone phosphate

Glyceraldehyde-3-phosphate

P-

-P

Glycolysis
Oxidation reactions 2 -P
Glyceraldehyde-3-phosphate

2 Pi
2 P* -P
1,3-bisphosphoglycerate

2 NAD+ 2 NADH 2 ADP 2 ATP

-P

3-phosphoglycerate

Glycolysis
Oxidation reactions 2 -P 3-phosphoglycerate

P
|

2-phosphoglycerate
2 H 2O

Phosphoenolpyruvate

2 ADP 2 ATP

PYRUVATE

Glycolysis
In preparatory reactions, two ATP molecules used In oxidation reactions, four ATP molecules made (net gain of two ATP molecules) and 2 molecules of NADH Fate of pyruvate: aerobic respiration, enters CAC (complete breakdown) anaerobic respiration (less energy produced) fermentation (converted to different end-products)

Respiration
Aerobic respiration Citric acid cycle (CAC) (also called Krebs cycle, TCA cycle): series of biochemical reactions resulting in the oxidation of acetyl coenzyme A (derived from pyruvate) to NADH, FADH2 and some ATP Pyruvate cannot enter CAC directly, is decarboxylated
CoA CO2

Pyruvate

Acetyl CoA
-CoA

NAD+

NADH

Respiration
Citric acid cycle

= carbon

Respiration
Electron transport system Sequence of membrane-associated electron carrier molecules capable of oxidation and reduction As electrons pass through system, energy released, drives generation of ATP Eukaryotes: located in inner membrane of mitochondria Prokaryotes: located in plasma membrane

Respiration
Electron transport system Three classes of carrier molecules: Flavoproteins (contain flavins, derived from vitamin B2, flavin mononucleotide [FMN] important) Cytochromes (proteins with iron group [heme] which exists as Fe2+ [reduced] or Fe3+ [oxidised]) Ubiquinones (co-enzyme Q, small non-protein carriers)

Respiration
Electron transport system Bacteria have diverse electron transport systems Types of carriers and order of function differs in different bacteria and from mitochondrial systems Basic function is the same: release energy (as electrons) from higher-energy compounds and transfer to lower-energy compounds

Respiration
Electron transport system (mitochondria) 6 H+

2 H+

2 H+
Q

2 H+
cyt b cyt c1
cyt c cyt a cyt a3

FMN

2 H+
NADH

+ H+

NAD+

2 H+
Cytochrome b-c1 complex

O2

H2 O
3 ADP + 3 Pi
3 ATP

NADH dehydrogenase complex

Cytochrome oxidase complex

ATP synthase

Respiration
Energy production in aerobic respiration:

Glycolysis

Glucose 2 Pyruvate 2 Acetyl CoA

NADH 2 2 6

FADH2

ATP 2

Citric acid cycle Electron transport

2 34 38

Respiration
Summary of aerobic respiration in eukaryotes:

C6H12O6 + 6 O2 + 38 ADP + 38 Pi
Glucose Oxygen

Water

Carbon dioxide

6 CO2 + 6 H2O + 38 ATP

In prokaryotes, aerobic ATP yields can be less because of truncated electron transport systems

Respiration
Electron transport system of E. coli

Respiration
Anaerobic respiration Final electron acceptor: inorganic substance other than O2 Pseudomonas, Bacillus and enteric bacteria (e.g. E. coli) can use nitrate ion (NO3-), reduced to nitrite (NO2-) Desulfovibrio: sulphate (SO42-) hydrogen sulphide (H2S) Others: carbonate (CO32-) methane (CH4), Fe3+ Fe2+ ATP yields not as high as aerobic respiration (only part of CAC used, not all carriers in electron transport participate); anaerobes grow more slowly than aerobes; allows growth when O2 absent

Respiration
Electron transport systems of E. coli during (a) aerobic and (b) anaerobic respiration [using NO3- as the final electron acceptor]

Respiration
Anaerobic respiration Electron transport systems contain cytochromes, quinones, iron-containing proteins; analogous to aerobic electron transport Some bacteria (facultative anaerobes) carry out aerobic respiration until O2 depleted, switch to anaerobic respiration Others (obligate anaerobes) cannot use O2 and can be killed by it

Fermentation
Does not require O2 (uses organic molecule as the final electron acceptor) Does not use CAC or electron transport system (no oxidative phosphorylation, only substrate-level) Provides small amount of ATP (glycolysis only), energy stored in bonds of end products

Purpose of fermentation: to oxidise NADH NAD+ (or NADPH NADP+); NAD+ returned to glycolysis (energy-producing stage)

Fermentation
Different end-products depend on microorganism, substrate and enzymes, therefore, analysis of endproducts used in identification of microorganisms

Fermentation
Two important fermentations: - lactic acid fermentation - alcohol fermentation

Lactic acid fermentation

2
Lactate dehydrogenase

Pyruvate 2 NADH 2 NAD+ + H+ Lactic acid

Fermentation
Homolactic (homofermentative) fermentation: only lactic acid produced by fermentation (Streptococcus, Lactobacillus) Heterolactic (heterofermentative) fermentation: fermentation produces lactic acid plus other acids or alcohols and CO2 (Leuconostoc)

Fermentation
Alcohol fermentation
2
Pyruvate decarboxylase

Pyruvate

2
Alcohol dehydrogenase

Acetaldehyde + CO2 2 NADH 2 NAD+ + H+ Ethanol

Lipid and protein catabolism

Glucose = main energy-supplying carbohydrate Microorganisms also oxidise lipids and proteins Oxidation of all these nutrients related Microorganisms break down lipids fatty acids and glycerol (lipases) and proteins amino acids (proteases, peptidases); these can enter the glycolytic pathway, CAC after appropriate conversion

Lipid and protein catabolism

Proteins Carbohydrates Sugars Glucose Glycerol DHAP * G-3-P Acetyl CoA Lipids Fatty acids Beta oxidation

Amino acids
Deamination Decarboxylation Dehydrogenation

Acetyl CoA

CAC

Electron transport

* Dihydroxyacetone phosphate

Metabolism of energy use (anabolism)

Uses of ATP: active transport of substances across membranes flagellar motion movement production of new cellular components (major use)

Metabolism of energy use (anabolism)

Autotrophs/lithotrophs fix CO2 organic compounds (Calvin cycle) Heterotrophs/organotrophs use available organic compounds (in chemoorganotrophs, used both as energy source and carbon source) Biosynthesis of: carbohydrates lipids amino acids nucleotides (purines and pyrimidines)

Polysaccharide biosynthesis
Microorganisms synthesise sugars and polysaccharides Glucose synthesised from intermediates of glycolysis and CAC Glucose and other simple sugars (hexoses) complex polysaccharides (e.g. glycogen) or cell wall components (e.g. peptidoglycan) Glucose phosphorylated (activated) and linked ATP or UTP are used as energy sources

Polysaccharide biosynthesis
Glucose
Glucose-6-phosphate
ATP

Fructose-6-phosphate
UTP

Adenosine diphosphoglucose

UDPG Glycogen Peptidoglycan LPS

Polysaccharides and nucleotide biosynthesis

(Hexose) (Pentose)
Glucose-6-phosphate

Ribulose-5-phosphate

+ CO2

Ribose-5-phosphate

Ribonucleotides Deoxyribonucleotides

RNA DNA

Biosynthesis of glucose (gluconeogenesis)

Other pathways

CAC

Oxalacetate
Phosphoenolpyruvate

+ CO2

Reversal of glycolytic steps

Glucose-6-phosphate

Biosynthesis of amino acids

Amino acids required for protein synthesis Some bacteria (e.g. E. coli) can make all amino acids from glucose and inorganic salts Others need to obtain some from the environment Citric acid cycle provides many of the precursors for amino acid synthesis; other precursors from glycolysis Addition of amine group to acid (amination) amino acid Transfer of amine group from one amino acid to another = transamination

Biosynthesis of amino acids

Biosynthesis of amino acids

Biosynthesis of amino acids

Amination:
NH2 -Ketoglutarate + NH3
Glutamate dehydrogenase

Glutamate

NH2 Glutamate + NH3

Glutamine synthetase

NH2

NH2

Glutamine

Biosynthesis of amino acids

Transamination:
NH2 Glutamate + Oxalacetate
Transaminase

NH2 -Ketoglutarate + Aspartate

Biosynthesis of fatty acids

Fatty acids required to make lipids in Bacteria and Eukarya Used in biological membranes; cholesterol (eukaryotes); waxes (acid-fast bacteria); pigments; chlorophyll; energy storage Lipids made by joining glycerol and fatty acids Glycerol derived from dihyroxyacetone phosphate (glycolysis intermediate) Fatty acids derived from acetyl CoA; successive addition of 2-carbon fragments

Biosynthesis of fatty acids

Glucose Glycolysis
Glyceraldehyde-3phosphate Dihydroxyacetone phosphate

Pyruvate

Glycerol Lipids

Acetyl CoA

Fatty acids

CAC

Interrelationship of metabolic pathways

Cell wall Cytoplasmic membrane
Polysaccharides Peptidoglycan

Phospholipids Glucose G-3-P Pyruvate

Amino acids

Ribose-P

Nucleotides

DNA

Fatty Acids

Acetyl-CoA
Amino acids

RNA

ENERGY

CAC

Proteins

Nutrition and growth (Brock Ch. 5 & 6)

Requirements for microbial growth - physical - chemical Culture media Growth of microorganisms - growth rates - phases of growth Measurement of microbial growth - direct methods - indirect methods

Nutrition and growth

Requirements for microbial growth
Physical temperature pH osmotic pressure Chemical oxygen sources of C, N, S, P, trace elements organic growth factors

Temperature
One of the most important environmental factors affecting microbial growth Most microorganisms grow well at temperatures favoured by humans Certain bacteria can grow at extreme temperatures Each species has particular minimum, optimal and maximum growth temperatures

Temperature

Temperature
Microorganisms classified into four broad groups: psychrophiles (low temperature optima) mesophiles (mid-range temperature optima) thermophiles (high temperature optima) hyperthermophiles (very high temperature optima)

Temperature

Temperature
Psychrophiles: optimal temp = 15C or lower maximum temp = <20C minimal temp = 0C or lower found in constantly cold environments (killed by brief exposure to room temperature) Examples: algae growing under polar ice, surface of snowfields

Temperature
Mesophiles: optimal temp = 25-40C maximum temp = <50C minimal temp = ~10C most of the common food spoilage and disease organisms, adapted to live in bodies of animals, temperature optimum close to body temperature of host

Temperature
Psychrotolerant microorganisms (psychrotrophs): temperature optimum of 20-40C, but can grow at 0C grow well at refrigeration temperatures responsible for food spoilage, food-borne disease 0C not optimal, spoil food over time

Temperature

Temperature

Temperature
Thermophiles: optimal temp = 50-60C maximum temp = <70C minimal temp = 45C grow at about temperature of hot water tap, sunlit soil, thermal vents (hot springs), important in compost (60-65C)

Temperature
Hyperthermophiles: optimal temp = >80C maximum temp = 113C minimal temp = 65C members of Bacteria and Archea, found in hot springs associated with volcanic activity, sulphur-utilising

Temperature
Molecular adaptations to extreme temperature:

Psychrophiles
enzymes have greater -helix content greater flexibility in cold more polar, less hydrophobic amino acids greater flexibility unsaturated (polyunsaturated) fatty acids in membranes active transport across membranes at low temperature (saturated = waxy, non-functional)

Temperature
Molecular adaptations to extreme temperature:

Thermophiles
critical amino acid differences in enzymes, salt bridges (ionic bonds) resist unfolding at high temperatures ribosomes are heat stable saturated fatty acids in membranes heat stable hyperthermophiles (Archea): lipid monolayer heat stable

Temperature
Molecular adaptations to extreme temperature:

Eukaryotes
none exist above 60C organelle membranes need to remain porous (passage of large molecules: ATP, RNA), not stable at high temperatures

Temperature
Biotechnology and thermophiles:
advantages for industrial and biotechnological processes enzymes from thermophiles catalyse reactions more rapidly and efficiently at higher temperatures, more stable (longer shelflife) Taq polymerase (Thermus aquaticus) used in polymerase chain reaction, not denatured by high temperatures used to melt DNA strands

pH
all microbes have a pH range and pH optimum microorganisms with pH optima = most natural environments (pH 5-9) are most common very few bacteria grow below pH 4; fermented foods have low pH (bacteria acid) = preservation some species <pH 2 or >pH 10 fungi are generally more acid tolerant than bacteria, pH 5 or below, some pH 2

pH
acidiphiles (extremophiles): live at low pH some Bacteria and Archea are obligate acidophiles Sulfolobus grows in drainage water of coal mines, oxidises S H2SO4; grows at pH 1 obligate acidophiles need high H+ ion concentration for membrane stability; neutral pH membrane dissolving

pH
alkaliphiles: pH optima of pH 10-11 found in highly basic habitats (soda lakes, high carbonate soils) some extreme alkaliphiles also halophiles (Archea)

pH
Extracellular pH vs intracellular pH
pH optimum = extracellular pH intracellular pH must remain near neutral (prevent destruction of acid or alkaline sensitive macromolecules) Extremophiles may have intracellular pH several units from neutral

pH
Buffers
when bacteria cultured in laboratory, often
produce acid, eventually interferes with growth

chemical buffers added to media to neutralise acids peptone phosphate buffers (KH2PO4) function near neutral pH (6-7.5), common for most bacteria, nontoxic, provide P (essential nutrient)

Osmotic pressure
microorganisms obtain almost all nutrients in solution from surrounding water are 80-90% water availability of water depends on water content of environment concentration of solutes (sugars, salts) solutes have affinity for water, makes it unavailable to microorganisms

Osmotic pressure
water availability = water activity (aw); range = 0-1, microbial activity between ~0.7-1.0, fungi < bacteria microbial cell in hypertonic solution (higher [solute] than inside cell), H2O passes out into solution plasmolysis (shrinkage of cytoplasmic membrane)

Osmotic pressure
Plasmolysis

Osmotic pressure
growth of cell inhibited as membrane pulls away from cell wall death or dehydration and dormancy addition of salts or sugars to solution or food lower aw, higher osmotic pressure; used to preserve foods salted fish, honey, condensed milk

Osmotic pressure
Water activity ranges for selected foods
1.00-0.95 eggs 0.95-0.90 orange 0.90-0.80 0.80-0.70 0.70-0.60 0.60-0.50 0.40 0.30 0.20 Fresh meat, fruit, vegetables, canned fruit in syrup, canned vegetables in brine, margarine, butter, Processed cheese, bakery goods, high moisture prunes, raw ham, dry sausage, high-salt bacon, juice concentrate Aged cheddar cheese, sweetened condensed milk, Hungarian salami, jams Molasses, soft dried figs, heavily salted fish Parmesan cheese, dried fruit, corn syrup, liquorice Chocolate, confectionery, honey, noodles Dried egg, cocoa Dried potato flakes, potato crisps, crackers, cake mixes, pecan halves, peanut butter Dried milk, dried vegetables, chopped walnuts

Osmotic pressure
Survival in environments of high osmotic pressure
halophiles: environments with high osmotic
pressure are mainly those with high [NaCl] (seawater); microbes found in the sea have requirement for salt and grow optimally at aw of seawater (0.98) mild halophiles = 1-6% NaCl moderate halophiles = 6-15% NaCl extreme halophiles = 15-30% NaCl (Dead Sea)

Osmotic pressure
Survival in environments of high osmotic pressure
halotolerant (facultative halophiles): can tolerate reduction in aw (up to 2% NaCl) osmophiles: grow in high [sugar] xerophiles: grow in very dry environments (lack of water)

Osmotic pressure
Survival in environments of high osmotic pressure
growth under high osmotic pressure possible because
cell increases internal solute concentration, H2O enters cell, adjusts cytoplasmic aw

pumping inorganic ions (K+) into cell, synthesising or concentrating organic solutes (amino acids, carbohydrates, alcohols) these are called compatible solutes (non-inhibitory to biochemical processes, H2O soluble)

Oxygen
micoorganisms classified according to oxygen requirement or tolerance aerobes: grow at full O2 tension (air = 21% O2), some tolerate hyperbaric levels microaerophiles: use O2 at levels lower than found in air (limited respiration, O2sensitive enzymes) facultative: either aerobic or anaerobic

Oxygen
anaerobes: lack an aerobic respiratory system, do not use O2 as terminal electron acceptor, carry out anaerobic respiration, grow in absence of O2 aerotolerant anaerobes: tolerate O2, can grow in presence but dont use obligate (strict) anaerobes: killed by O2

Oxygen
A = aerobe B = obligate anaerobe C = facultative anaerobe D = microaerophile E = aerotolerant anaerobe

Oxygen
Toxic forms of oxygen
normal ground state oxygen toxic forms of oxygen: 1. Singlet oxygen: O2 boosted to higherenergy state photochemically or biochemically; extremely reactive; caroteniods (pigments) convert to non-toxic forms

Oxygen
Toxic forms of oxygen
other toxic forms are by-products of reduction of O2 H2O in respiration:
2. Superoxide anion (O2-): O2 + e- O2- ; very toxic because very unstable, steal electrons from other molecules, these in turn steal electrons, etc.; superoxide dismutase (SOD) neutralises; aerobes, facultative anaerobes, aerotolerant anaerobes produce SOD [ O2- + O2- H2 O 2 + O2 ]

Oxygen
Toxic forms of oxygen
3. Hydrogen peroxide: can damage cell components, not as toxic as superoxide or hydroxyl radical; catalase [ 2H2O2 2H2O + O2 ] or peroxidase [ H2O2 + 2H+ 2H2O ] neutralise hydrogen peroxide 4. Hydroxyl radical: most reactive, instantly oxidises any organic substance in cell; produced from hydrogen peroxide [ H2O2 + e- + H+ H2O + OH ]

Oxygen
Toxic forms of oxygen
Peroxidase SOD

H2 O

O 2Superoxide

O2 + H2O2 H2 O + O 2

Catalase

Oxygen
Toxic forms of oxygen
obligate anaerobes are extremely sensitive to O2
obligate anaerobes produce neither SOD or catalase leads to accumulation of superoxide anions in cytoplasm aerotolerant anaerobes produce SOD or equivalent microaerophiles produce superoxide anions and H2O2 in [lethal] in O2-rich conditions

Oxygen
Toxic forms of oxygen
_________________________________________________________ Group SOD Catalase Peroxidase _________________________________________________________ Obligate aerobes and most facultative anaerobes (e.g. Enterics) Most aerotolerant anaerobes (e.g. Streptococci) + + + +

Obligate anaerobes (e.g. Clostridia) _________________________________________________________ -

Oxygen
Toxic forms of oxygen
laboratory culture of obligate anaerobes: reducing media - contains ingredients (sodium thioglycollate) that combine with dissolved O2 and deplete from media, used in tubes, heated before use to drive off absorbed O2 culture grown on Petri dish to observe individual colonies requires specialised techniques

Oxygen
Toxic forms of oxygen
laboratory culture of obligate anaerobes: anaerobic jars - O2 removed by adding H2O to packet of sodium bicarbonate and sodium borohydrate; H2 and CO2 produced; palladium catalyst in jar combines O2 with H2 H2O; O2 quickly disappears; CO2 helps growth of anaerobes

Oxygen
Toxic forms of oxygen
laboratory culture of obligate anaerobes: anaerobic chambers - transparent chamber fitted with air locks; filled with inert gases; airtight rubber gloves (glove ports) fitted to wall of chamber; hands inserted into gloves allows manipulation inside chamber.

Oxygen

Anaerobic jar

Anaerobic chamber

Oxygen
Capnophiles
aerobes that grow better at higher [CO2] than present in air candle jar: lighted candle placed in sealed jar with
cultures; candles stops burning when [O2] levels fall; [CO2] elevated

CO2 incubators: electronic control of [CO2] commercial packets: contain CO2 generator; tube crushed, chemicals mixed, reaction produces CO2 to 10%; O2 reduced to 5%

Carbon
one of the most important requirements for microbial growth (along with water) structural backbone of living matter needed for all organic compounds half of dry weight of cell is carbon carbon obtained from energy source carbohydrate, protein, lipids (chemoorganotrophs), organic compounds (photoheterotrophs), or from CO2 (chemolithotrophs, photoautotrophs)

Other elements - N, S and P

needed for synthesis of cellular material
proteins require N and S DNA, RNA, ATP require N and P N = 12% dry weight P and S = 4% dry weight N obtained from amino group of amino acids; proteins decomposed; amino acids incorporated into new proteins and other compounds

Other elements - N, S and P

other bacteria obtain N from NH4+ in cellular material or nitrates (NO3-) nitrogen fixers: use gaseous N2; important process (nitrogen fixation); free-living (photosynthetic cyanobacteria) or symbiotic (Rhizobium and legumes), nitrogen used by bacterium and plant sulphur used in S-containing amino acids and vitamins (thiamine, biotin); sulphate (SO42-), H2S and amino acids are sources of sulphur

Other elements - N, S and P

phosphorus essential for nucleic acids and phospholipids (cytoplasmic membranes) high energy bonds of ATP phosphate (PO43-) important source of P

Other elements
other elements required by microorganisms (cofactors for enzymes) K: required by protein synthesis enzymes Mg: stabilises ribosomes, required for activity of many enzymes Ca: stabilises cell wall, heat stability of endospores Na: requirement depends of habitat (seawater vs freshwater) Fe: key component of cytochromes (electron transport), siderophores are iron-binding agents that transport Fe into cell

Trace elements
required in very small amounts (micronutrients) metals (e.g. Mn, Mo, Ni, Zn) structural role in enzymes naturally present in water, even distilled water, and other media components, therefore not normally added to laboratory media

Organic growth factors

essential organic compounds unable to be synthesised (some vitamins in humans) directly obtained from environment e.g. vitamins (co-enzymes), amino acids, purines, pyrimidines most bacteria can make all vitamins, some cannot; those unable to be made are organic growth factors

Culture media
nutrient material prepared for growth of microorganisms in the laboratory some microorganisns cannot be grown on synthetic media; need living host Mycobacterium leprae grown in armadillos obligate intracellular bacteria [rickettsias, chlamydias] and viruses reproduce only in living cells

Culture media
criteria met by culture medium correct nutrients for particular microorganism sufficient moisture properly adjusted pH sufficient level of O2 (sometimes none!) sterile correct incubation temperature

Culture media
growth on solid media requires the addition of agar (complex carbohydrate derived from marine algae) agar acts as a solidifying agent useful properties of agar few microbes can degrade liquifies at 100C, gels at 40C

Culture media
Chemically defined media: exact chemical composition is known precise amounts of purified chemicals added to dH2O Complex undefined (complex) media: use digests of proteins (peptones), casein (milk protein), beef, soybeans, yeast cells dissolved in dH2O highly nutritious but chemically undefined nutrient broth (liquid) or agar (solid) basal or enriched (fastidious organisms)

Culture media
selective and differential media, used in clinical and public health microbiology selective media: suppress the growth of unwanted bacteria encourage growth of desired ones e.g. Bismuth sulphite agar - selective for Salmonella - bismuth sulphite inhibits Gram positive bacteria and most other Gram negative bacteria

Culture media
differential media allow desired microorganisms to be distinguished from others e.g. blood agar (contains red blood cells: horse, sheep) differences in lytic reactions identification of streptococci: -haemolysis - green or brownish halo -haemolysis - zone of complete haemolysis -haemolysis - no haemolysis

Culture media

-haemolysis

-haemolysis

-haemolysis

Culture media
selective and differential media combined Mannitol Salt Agar (isolation of Staphylococcus aureus) 7.5% NaCl (selective for staphyloccoci) mannitol and pH indicator (S. aureus ferments mannitol acid pH change (bright yellow colour) MacConkey Agar (isolation of enteric bacteria) bile salts [and crystal violet] (inhibits Gram positive bacteria and non-enterics) lactose and pH indicator (differentiates fermenters [red/pink] from non-fermenters)

Culture media
enrichment culture used to when desired bacteria present in small numbers, other bacteria in larger numbers designed to increase very small numbers of desired organisms to detectable levels often used for soil or faecal samples medium and incubation conditions selective for desired organisms, counter-selective for others

Pure cultures
infectious materials, environmental, food samples contain many different microorganisms plating on solid medium visible colonies one cell or spore one colony distinctive colony morphology allows identification experimentation and testing requires pure cultures; derived from single colony, clones

Pure cultures
streak plate method objective: to separate cells in inoculum into individual cells individual colonies if desired microorganism is not present in large numbers, selective enrichment occurs prior to isolation by streak plate method

Pure cultures
streak plate method
Flame

Flame
E C

Flame

Flame

Pure cultures
streak plate method

Growth of microorganisms
growth = increase in number of cells = increase in microbial mass increase in size of individual cell is insignificant cell division by binary fission (one cell two); budding, fragmentation all cell constituents (macromolecules, monomers, inorganic ions) increase in number cell elongates, partition (septum) forms, daughter cells pinched off, cells separate

Growth of microorganisms

One generation

Growth of microorganisms
growth rate = cell number (cell mass) / unit time generation time = time required for cell to divide and population to double (doubling time) generation times: 1-3 hours (bacteria) extremes = 10 minutes several hrs days E. coli = 20-30 minutes

Growth of microorganisms
_____________________________________
Generation number

_____________________________________
0 1 2 3 4 5 10 15 20 1 (= 20) 2 (= 21) 4 (= 22) 8 (= 23) 16 (= 24) 32 (= 25) 1,024 (= 210) 32,768 (= 215) 1,048,576 (= 220) 0 0.301 0.602 0.903 1.204 1.505 3.01 4.515 6.021

Number of cells Arithmetic Logarithmic (log10)

_____________________________________

Growth of microorganisms
Arithmetic number of cells
7 1200000 1000000 800000 600000 400000 200000 0 0 5 10 Generations 15 20 Arithmetic Logarithmic 6 5 4 3 2 1 0

Logarithmic (log1 0) number of cells

Growth of microorganisms
exponential growth: population increase where cell number doubles per unit time calculating generation times 21 22, 22 23, 23 24 ... N = N0 2 n N = final cell number N0 = initial cell number n = number of generations

Growth of microorganisms
N = N0 2 n n = log N - log N0 log 2 = log N - log N0 0.301 = 3.3 (logN - logN0)

Growth of microorganisms
g (generation time) = t (time) n N = 2.5x107, N0 = 103, t = 8 hours, g = ? n = 3.3 (log [2.5x107] - log [103]) = 3.3 (7.39 - 3) = 3.3 x 4.39 = 14.5 g = 480 = 33 minutes 14.5

Growth of microorganisms
Four phases of bacterial growth (in liquid culture) Lag phase period of little or no cell division (1 hour several days) cells do not immediately reproduce not dormant, intense period of metabolic activity (DNA, enzyme synthesis) lag phase not always seen (exponential phase culture same medium, same conditions) lag: cells transferred from rich medium to poorer one; damage to cells before culture

Growth of microorganisms
Exponential (Log) phase healthiest state of cells one cell becomes two rates of exponential growth vary between microbes (= slope on graph); influenced by growth conditions (temperature, nutrients) Stationary Phase exponential growth cannot occur indefinitely essential nutrients used up waste products build up

Growth of microorganisms
no net increase or decrease in cell number cryptic growth (cells continue to carry out some metabolic activities) Death Phase rate of cell death increases exponential rate of decline, usually slower than exponential growth tiny fraction of cells remain, or population dies completely can take a few days, some microbes can retain some surviving cells indefinitely

Growth of microorganisms

Phases relate to populations of cells, not individual cells

Measurement of growth
Population growth = changes in cell numbers or weight of cell mass Methods for estimating cell numbers or mass direct methods direct microscopic count most probable number viable (plate, colony) counts indirect methods turbidimetric measurement metabolic activity dry weight

Direct microscopic count

Breed count method (used to count bacteria in milk): measured volume (0.01 ml) of bacteria suspension
placed in defined area (1 cm2) of slide stain added to visualise cells area of viewing field determined number of bacteria counted (average of several fields) number of bacteria in suspension determined

Direct microscopic count

area of viewing field determined by using stage micrometer (0.01 mm divisions) 16 divisions seen, diameter = 0.16 mm, radius = 0.08 mm A = r2 = 3.14 x 0.082 = 0.02 mm2 Microscope factor = 100/A = 100/0.02 = 5000 Average of 30 cells/field, 30 x 5000 = 1.5x105 cm2 cells/

Original sample was 0.01 ml; therefore, X 100 to convert to ml, i.e. 1.5 x 105 x 100 = 1.5 x 107 cells/ml

Direct microscopic count

Stage micrometer: each division = 0.01 mm

Direct microscopic count

Haemocytometer (Petroff-Hauser cell counter)

Direct microscopic count

Haemocytometer (Petroff-Hauser cell counter) 12 cells / 0.04 mm2 (1/25 mm2) 12 cells / 0.0008 mm3 (depth of chamber = 0.02 mm) = 15000 cells / mm3 = 15000 x 1000 cells / cm3 (ml) = 15000000 cells / ml = 1.5 x 107 cells / ml

Direct microscopic count

Limitations: cannot distinguish dead cells from live cells small cells difficult to see imprecise staining to visualise cells (phase contrast if unstained) not suitable for low cell numbers (<106 / ml)

Most probable number method

statistical estimating technique premise: more bacteria in a sample, more dilution required to reduce density to point where no cells left to grow in a series of tubes MPN tables consulted, used to determine the numbers likely to give observed result (refer to prac notes)

Viable counts
most frequently used method measures number of viable cells assumes that one cell will yield one colony; some bacteria linked in chains or clumps, colony derived from >1 cell results expressed as colony forming units (cfu) results can take up to 24 hours, a problem in some situations (food micro)

Viable counts
Two methods: spread plate: volume (0.1 ml) spread over surface of agar plate; volumes >0.1 ml avoided, excess liquid can make colonies coalesce, difficult to count pour plate: volume (0.1-1 ml) added to sterile Petri dish, molten agar added, mixed and allowed to set; colonies grow on surface and subsurface; larger volumes can be used, organism must be able to withstand 45C (molten agar)

Viable counts

Spread plate

Pour plate

Viable counts
Serial dilutions: for both methods, number of colonies cannot be too large (overcrowding: not all cells colonies, fusion of colonies counting errors) or too small (statistical significance of counting) only plates with 30-300 colonies are counted appropriate numbers obtained by serial dilution several 10-fold dilutions of sample (sometimes 100-fold)

Viable counts

10-1

10-2 10-3

10-4

10-5

10-6

Indirect methods
Turbidimetric measurement of cell number bacterial suspension looks cloudy (turbid), light scattered: more cells, more turbid, more light scattered turbidity measured by spectrophotometer: pass light through suspension, measure unscattered emergent light absorbance or optical density (OD) = log10 (1/T) (T = percentage of transmission) OD proportional to cell number

Indirect methods

100

Light source

Blank

Spectrophotometer

100

Bacterial suspension

Indirect methods
Measurement of metabolic activity of population assumes that amount of a metabolic product (acid, CO2) is proportional to cell number Dry weight useful for filamentous organisms (moulds) where discrete colonies not formed organism removed from growth medium, filtered, dried in desiccator, weighed