topic 2_2012
TRANSCRIPT
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Topic 2
Microbial metabolism (Ch. 4)
Nutrition and growth (Ch. 5)
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Microbial metabolism
Metabolism = sum of all chemical reactions in aliving organism
All chemical reactions release or require energy
Metabolism = energy balancing act
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Microbial metabolism
Catabolism: release energy, breakdown ofcomplex organic compounds simplerones, hydrolytic (use H2O, chemical bondsbroken), exergonic (net energyproduction)
Anabolism: require energy, building of complex
organic molecules from simpler ones,dehydration reactions (release H2O),endergonic (net energy consumption),biosynthetic
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Microbial metabolism
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Interrelationship of metabolic pathways
Glucose
Pyruvate
Acetyl-CoA
CAC
Fatty Acids
G-3-P
Phospholipids
Cytoplasmicmembrane
Polysaccharides Peptidoglycan
Cell wall
Amino acids
Ribose-P
Nucleotides DNA
RNA
Amino acids
ProteinsENERGY
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Microbial metabolism
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Energy production
Anabolic reactions:
ATP ADP + Pi + energy
Catabolic reactions:
ADP + Pi + energy ATP
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Energy stored in the chemical bonds of ATP
High energy bonds = unstable bonds
Energy released quickly and easily
Used for anabolic reactions
Energy production
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How much energy is needed by E. coli?
Cell constituent Number of Molecules synthesised Molecules of ATPmolecules per cell per second required per second
for synthesis
DNA 1 0.00037 60,000RNA 15,000 12.5 75,000Polysaccharides 39,000 32.5 65,000Lipids 15,000,000 12,500 87,000Proteins 1,700,000 1,400 2,120,000
Synthesis of a single DNA molecule (i.e. one replication)will consume approximately 162,000,000 ATP molecules
Energy production
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Energy produced in two general ways
Oxidation-reductionATP generation
Oxidation = removal of electrons from atom ormolecule, often produces energy
Reduction is the gain of one or more electronsOxidation and reduction are coupled reactions, one
molecule is oxidised, another is reduced = redoxreactions
Energy production
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Cellular oxidation involves simultaneous loss of electron andproton (H+ and e-), electrons cannot exist free insolution; loss of H atoms = hydrogenation
Energy production
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Co-enzymes help in the oxidation of organic substrates byaccepting protons (other co-enzymes donateelectrons)
Nicotinamide adenine dinucleotide (NAD+)
Nicotinamide adenine dinucleotide phosphate (NADP+)= important cellular co-enzymes (derived fromvitamins, B vitamins)
NAD+
primarily involved in catabolic reactionsNADP+ primarily involved in anabolic reactions
Energy production
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Energy production
Organic molecule Donates two H atoms(2 X [H+ + e-])
NAD+
+
Oxidised organic molecule
NADH + H++
NAH+ receivesone H atom and one e-NADH contains moreenergy, used to generateATP in later reactions
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Biological oxidation-reduction degrades highly reducedcompounds (nutrient molecules with many Hatoms) 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
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Substrate-level phosphorylation:High-energy Pi directly transferred from phosphorylatedcompound 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 transportsystem) in plasma membrane (prokaryotes) orinner mitochondrial membrane (eukaryotes)
Electron transfer releases energy, generates ATP
Energy production
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Photophosphorylation:Occurs in photosynthetic cells, light-trapping pigments(chlorophyll)
Organic molecules (sugars) synthesised with energyfrom light using CO
2and H
2O
Light energy converted to chemical energy (ATP andNADPH), used to synthesise organic molecules
Involves electron transport system
Energy production
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Carbohydrates are primary source of energy in mostmicroorganisms
Breakdown of carbohydrate molecules to produce energyis of great importance in cell metabolism
Glucose is the most common carbohydrate energy source
Microorganisms can also catabolise lipids and proteins
energy
Carbohydrate catabolism
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Microorganisms derive energy from carbohydrates by three
processes:1. Aerobic respiration2. Anaerobic respiration3. Fermentation (anaerobic)
Carbohydrate catabolism
(Organiccompounds)
Glucose Pyruvate
Citric acidcycle
ElectronTransport (O2)
[Citric acidCycle]
ElectronTransport(Not O2)
1
2
3Energy
(Terminal electronacceptors)
1 = +++2 = ++3 = +
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Both respiration and fermentation begin with glycolysis(oxidation of glucose to pyruvate, some ATP andNADH 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 FADH2oxidised, cascade of redox reactions energy, generates considerable amount of ATP
Carbohydrate catabolism
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Fermentation:No CAC or electron transport, much lower ATP yieldPyruvate converted to different products, depending
on microorganism (e.g. alcohol, lactic acid,
other acids)Occurs in absence of oxygen (anaerobic)
Carbohydrate catabolism
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Also called the Emden-Myerhoff pathwayEnzymes catalyse the splitting of glucose (6-carbon
sugar) into two 3-carbon sugars
The 3-carbon sugars are oxidised 2X pyruvateNAD+ reduced to NADH, net production of two ATP
molecules (substrate level phosphorylation)
Can occur in presence or absence of oxygenTwo stages: preparatory reactions and oxidation
(energy-conserving) reactions
Glycolysis
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Preparatory reactions
Glycolysis
Glucose
Glucose-6-phosphate
ATP
ADP
Fructose-6-phosphate
-P
-P
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Preparatory reactions
Glycolysis
ATP
ADP
Fructose-6-phosphate
Fructose-1,6-phosphate
Dihydroxyacetone phosphate Glyceraldehyde-3-phosphate
-P
-PP-
P- -P
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Oxidation reactions
Glycolysis
1,3-bisphosphoglycerate
3-phosphoglycerate
Glyceraldehyde-3-phosphate2
2 NAD+
2 NADH2
2 Pi
2
2 ATP
2 ADP
-P
-PP*
-P
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In preparatory reactions, two ATP molecules used
In oxidation reactions, four ATP molecules made (net gainof 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)
Glycolysis
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Aerobic respiration
Citric acid cycle (CAC) (also called Krebs cycle, TCAcycle): series of biochemical reactions resulting in the
oxidation of acetyl coenzyme A (derived frompyruvate) to NADH, FADH2 and some ATP
Pyruvate cannot enter CAC directly, is decarboxylated
Pyruvate Acetyl CoA
Respiration
CoA CO2
NAD+ NADH
-CoA
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Respiration
Citric acid cycle
= carbon
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Electron transport system
Sequence of membrane-associated electron carriermolecules capable of oxidation and reduction
As electrons pass through system, energy released,drives generation of ATP
Eukaryotes: located in inner membrane ofmitochondria
Prokaryotes: located in plasma membrane
Respiration
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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-proteincarriers)
Respiration
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Electron transport system
Bacteria have diverse electron transport systems
Types of carriers and order of function differs indifferent bacteria and from mitochondrial systems
Basic function is the same: release energy (aselectrons) from higher-energy compounds and
transfer to lower-energy compounds
Respiration
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Respiration
NADH NAD+
NADH dehydrogenasecomplex
FMN
2 H+
+ H+
Q
Cytochrome b-c1complex
cyt b
cyt c1
cyt c
2 H+
2 H+2 H+
2 H+
cyt a
cyt a3
O2 H2O
Cytochrome oxidasecomplex
ATPsynthase
6 H+
3 ATP3 ADP+ 3 Pi
Electron transport system (mitochondria)
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Energy production in aerobic respiration:
NADH FADH2 ATPGlycolysis Glucose
2 Pyruvate 2 2
2 Acetyl CoA 2
Citric acid cycle 6 2 2
Electron transport 34
38
Respiration
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Summary of aerobic respiration in eukaryotes:
C6H12O6 + 6 O2 + 38 ADP + 38 Pi Glucose Oxygen
6 CO2 + 6 H2O + 38 ATPCarbon dioxide Water
In prokaryotes, aerobic ATP yields can be less becauseof truncated electron transport systems
Respiration
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Electron transport system of E. coli
Respiration
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Anaerobic respiration
Final electron acceptor: inorganic substance other than O2Pseudomonas, Bacillusand enteric bacteria (e.g. E. coli) can
use nitrate ion (NO3-), reduced to nitrite (NO2-)
Desulfovibrio: sulphate (SO42-) hydrogen sulphide (H2S)Others: carbonate (CO
3
2-) methane (CH4), Fe3+ Fe2+
ATP yields not as high as aerobic respiration (only part ofCAC used, not all carriers in electron transportparticipate); anaerobes grow more slowly than
aerobes; allows growth when O2 absent
Respiration
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Electron transport systems of E. coliduring (a) aerobic
and (b) anaerobic respiration [using NO3- as thefinal electron acceptor]
Respiration
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Anaerobic respiration
Electron transport systems contain cytochromes,quinones, iron-containing proteins; analogous toaerobic electron transport
Some bacteria (facultative anaerobes) carry out aerobicrespiration until O2 depleted, switch to anaerobicrespiration
Others (obligate anaerobes) cannot use O2 and can bekilled by it
Respiration
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Does not require O2 (uses organic molecule as the finalelectron acceptor)
Does not use CAC or electron transport system (nooxidative phosphorylation, only substrate-level)
Provides small amount of ATP (glycolysis only), energystored in bonds of end products
Purpose of fermentation:to oxidise NADH NAD+ (or NADPH NADP+);NAD+ returned to glycolysis (energy-producingstage)
Fermentation
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Different end-products depend on microorganism,substrate and enzymes, therefore, analysis of end-
products used in identification of microorganisms
Fermentation
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Two important fermentations:- lactic acid fermentation
- alcohol fermentation
Lactic acid fermentation
2 Pyruvate
Lactate
dehydrogenase
2 Lactic acid
Fermentation
2 NADH
2 NAD+ + H+
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Homolactic (homofermentative) fermentation:
only lactic acid produced by fermentation(Streptococcus, Lactobacillus)
Heterolactic (heterofermentative) fermentation:
fermentation produces lactic acid plus other acids oralcohols and CO2 (Leuconostoc)
Fermentation
F
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Alcohol fermentation
2 Pyruvate
Pyruvate
decarboxylase
2 Acetaldehyde + CO2
Alcohol
dehydrogenase
2 Ethanol
Fermentation
2 NADH
2 NAD+ + H+
L d d b l
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Lipid and protein catabolism
Glucose = main energy-supplying carbohydrate
Microorganisms also oxidise lipids and proteinsOxidation of all these nutrients relatedMicroorganisms break down lipids fatty acids and
glycerol (lipases) and proteins amino acids(proteases, peptidases); these can enter the
glycolytic pathway, CAC after appropriateconversion
Li id d i b li
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Lipid and protein catabolism
Carbohydrates
Sugars
Glucose
Acetyl CoA
CAC
Proteins
Amino acids
DeaminationDecarboxylationDehydrogenation
Lipids
Glycerol Fatty acids
DHAP *
G-3-P
Acetyl CoA
Beta
oxidation
* Dihydroxyacetone phosphate
Electron transport
M b li f ( b li )
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Metabolism of energy use (anabolism)
Uses of ATP:
active transport of substances across membranesflagellar motion movement
production of new cellular components (major use)
M t b li f ( b li )
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Metabolism of energy use (anabolism)
Autotrophs/lithotrophs fix CO2 organic compounds(Calvin cycle)
Heterotrophs/organotrophs use available organic
compounds (in chemoorganotrophs, used both asenergy source and carbon source)
Biosynthesis of:carbohydrates lipidsamino acidsnucleotides (purines and pyrimidines)
P l h id bi th i
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Polysaccharide biosynthesis
Microorganisms synthesise sugars and polysaccharides
Glucose synthesised from intermediates of glycolysisand CAC
Glucose and other simple sugars (hexoses) complexpolysaccharides (e.g. glycogen) or cell wallcomponents (e.g. peptidoglycan)
Glucose phosphorylated (activated) and linkedATP or UTP are used as energy sources
P l h id bi th i
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Polysaccharide biosynthesis
Glucose
Glucose-6-phosphate
Adenosine diphosphoglucose
Glycogen
Fructose-6-phosphate
UDPG
UTP
Peptidoglycan LPS
ATP
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Bi th i f l ( l i )
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Biosynthesis of glucose (gluconeogenesis)
Glucose-6-phosphate
+ CO2
CAC
Other pathways
Oxalacetate
Phosphoenolpyruvate
Reversal of glycolytic steps
Bi th i f i id
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Biosynthesis of amino acids
Amino acids required for protein synthesisSome bacteria (e.g. E. coli) can make all amino acids from
glucose and inorganic salts
Others need to obtain some from the environmentCitric acid cycle provides many of the precursors for
amino acid synthesis; other precursors from glycolysis
Addition of amine group to acid (amination) amino acidTransfer of amine group from one amino acid to another =
transamination
Bi s th sis f mi ids
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Biosynthesis of amino acids
Bi s nth sis f min ids
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Biosynthesis of amino acids
Biosynthesis of amino acids
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Biosynthesis of amino acids
Amination:
-Ketoglutarate + NH3 GlutamateGlutamate
dehydrogenase
Glutamate + NH3 GlutamineGlutamine
synthetase
NH2
NH2 NH2 NH2
Biosynthesis of amino acids
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Biosynthesis of amino acids
Transamination:
Glutamate + OxalacetateTransaminase
-Ketoglutarate + Aspartate
NH2
NH2
Biosynthesis of fatty acids
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Biosynthesis of fatty acids
Fatty acids required to make lipids in Bacteria andEukaryaUsed in biological membranes; cholesterol (eukaryotes);
waxes (acid-fast bacteria); pigments; chlorophyll;energy storage
Lipids made by joining glycerol and fatty acidsGlycerol derived from dihyroxyacetone phosphate
(glycolysis intermediate)
Fatty acids derived from acetyl CoA; successiveaddition of 2-carbon fragments
Biosynthesis of fatty acids
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Biosynthesis of fatty acids
Glucose
Glyceraldehyde-3-phosphate
Acetyl CoA
CAC
G
lycolysis
Dihydroxyacetonephosphate
GlycerolPyruvate
Fatty acids
Lipids
Interrelationship of metabolic pathways
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Interrelationship of metabolic pathways
Glucose
Pyruvate
Acetyl-CoA
CAC
Fatty Acids
G-3-P
Phospholipids
Cytoplasmic
membrane Polysaccharides Peptidoglycan
Cell wall
Amino acids
Ribose-P
Nucleotides DNA
RNA
Amino acids
ProteinsENERGY
Nutrition and growth (Brock Ch 5 & 6)
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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
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Nutrition and growth
Requirements for microbial growth
PhysicaltemperaturepHosmotic pressure
Chemicaloxygensources of C, N, S, P, trace elementsorganic growth factors
Temperature
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Temperature
One of the most important environmentalfactors affecting microbial growth
Most microorganisms grow well attemperatures favoured by humans
Certain bacteria can grow at extremetemperatures
Each species has particular minimum, optimaland maximum growth temperatures
Temperature
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Temperature
Temperature
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Temperature
Microorganisms classified into four broadgroups:
psychrophiles (low temperature optima)mesophiles (mid-range temperature optima)thermophiles (high temperature optima)hyperthermophiles (very high temperature
optima)
Temperature
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Temperature
Temperature
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Temperature
Psychrophiles:
optimal temp = 15C or lowermaximum temp =
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Temperature
Mesophiles:
optimal temp = 25-40Cmaximum temp =
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Temperature
Psychrotolerant microorganisms(psychrotrophs):
temperature optimum of 20-40C, but cangrow at 0C
grow well at refrigeration temperaturesresponsible for food spoilage, food-borne
disease
0C not optimal, spoil food over time
Temperature
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Temperature
Temperature
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Temperature
Temperature
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Temperature
Thermophiles:
optimal temp = 50-60Cmaximum temp =
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Temperature
Hyperthermophiles:optimal temp = >80Cmaximum temp = 113Cminimal temp = 65Cmembers of Bacteria and Archea, found in hot
springs associated with volcanic activity,
sulphur-utilising
Temperature
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Temperature
Molecular adaptations to extreme temperature:Psychrophiles
enzymes have greater -helix content greater flexibility in coldmore polar, less hydrophobic amino acids
greater flexibility
unsaturated (polyunsaturated) fatty acids inmembranes active transport acrossmembranes at low temperature (saturated= waxy, non-functional)
Temperature
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Temperature
Molecular adaptations to extreme temperature:Thermophiles
critical amino acid differences in enzymes, saltbridges (ionic bonds) resist unfolding athigh temperatures
ribosomes are heat stablesaturated fatty acids in membranes heat
stablehyperthermophiles (Archea): lipid monolayer
heat stable
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Temperature
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Temperature
Biotechnology and thermophiles:
advantages for industrial and biotechnologicalprocesses
enzymes from thermophiles catalyse reactionsmore rapidly and efficiently at highertemperatures, more stable (longer shelf-life)
Taqpolymerase (Thermus aquaticus) used inpolymerase chain reaction, not denatured
by high temperatures used to melt DNAstrands
pH
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pH
all microbes have a pH rangeand pH optimummicroorganisms with pH optima = most natural
environments (pH 5-9) are most common
very few bacteria grow below pH 4; fermentedfoods have low pH (bacteria acid) =preservation
some species pH 10fungi are generally more acid tolerant than
bacteria, pH 5 or below, some pH 2
pH
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pH
acidiphiles (extremophiles): live at low pH
some Bacteria and Archea are obligateacidophiles
Sulfolobusgrows in drainage water of coalmines, oxidises S H2SO4; grows at pH 1
obligate acidophiles need high H+ ionconcentration for membrane stability;neutral pH membrane dissolving
pH
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pH
alkaliphiles: pH optima of pH 10-11
found in highly basic habitats (soda lakes, highcarbonate soils)
some extreme alkaliphiles also halophiles(Archea)
pH
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pH
Extracellular pH vs intracellular pHpH optimum = extracellular pH intracellular pH must remain near neutral(prevent destruction of acid or alkaline
sensitive macromolecules)
Extremophiles may have intracellular pHseveral units from neutral
pH
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p
Buffers
when bacteria cultured in laboratory, oftenproduce acid, eventually interferes withgrowth
chemical buffers added to media to neutraliseacids
peptonephosphate buffers (KH2PO4) function near neutral
pH (6-7.5), common for most bacteria, non-
toxic, provide P (essential nutrient)
Osmotic pressure
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p
microorganisms obtain almost all nutrients insolution from surrounding water
are 80-90% wateravailability of water depends on
water content of environmentconcentration of solutes (sugars, salts)
solutes have affinity for water, makes itunavailable to microorganisms
Osmotic pressure
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p
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 outinto solution plasmolysis (shrinkage ofcytoplasmic membrane)
Osmotic pressure
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p
Plasmolysis
Osmotic pressure
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p
growth of cell inhibited as membrane pullsaway from cell wall death or dehydration
and dormancy
addition of salts or sugars to solution or food lower aw, higher osmotic pressure; usedto preserve foods
salted fish, honey, condensed milk
Osmotic pressure
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p
Water activity ranges for selected foods
1.00-0.95 Fresh meat, fruit, vegetables, canned fruit in syrup,canned vegetables in brine, margarine, butter,
eggs0.95-0.90 Processed cheese, bakery goods, high moisture
prunes, raw ham, dry sausage, high-salt bacon,orange juice concentrate0.90-0.80 Aged cheddar cheese, sweetened condensed milk,
Hungarian salami, jams0.80-0.70 Molasses, soft dried figs, heavily salted fish0.70-0.60 Parmesan cheese, dried fruit, corn syrup, liquorice
0.60-0.50 Chocolate, confectionery, honey, noodles0.40 Dried egg, cocoa0.30 Dried potato flakes, potato crisps, crackers, cake
mixes, pecan halves, peanut butter0.20 Dried milk, dried vegetables, chopped walnuts
Osmotic pressure
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p
Survival in environments of high osmotic pressure
halophiles: environments with high osmoticpressure are mainly those with high [NaCl]
(seawater); microbes found in the sea haverequirement for salt and grow optimally ataw of seawater (0.98)
mild halophiles = 1-6% NaClmoderate halophiles = 6-15% NaCl
extreme halophiles = 15-30% NaCl(Dead Sea)
Osmotic pressure
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p
Survival in environments of high osmotic pressurehalotolerant (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
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p
Survival in environments of high osmotic pressuregrowth under high osmotic pressure possible because
cell increases internal solute concentration, H2Oenters cell, adjusts cytoplasmic a
w
pumping inorganic ions (K+) into cell, synthesising orconcentrating organic solutes (amino acids,carbohydrates, alcohols)
these are called compatible solutes (non-inhibitory tobiochemical processes, H2O soluble)
Oxygen
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yg
micoorganisms classified according to oxygenrequirement or tolerance
aerobes: grow at full O2 tension (air = 21%O2), some tolerate hyperbaric levels
microaerophiles: use O2 at levels lower thanfound in air (limited respiration, O2-
sensitive enzymes)
facultative: either aerobic or anaerobic
Oxygen
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yg
anaerobes: lack an aerobic respiratorysystem, 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
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A B C D E
A = aerobe
B = obligate anaerobe
C = facultative anaerobe
D = microaerophile
E = aerotolerant anaerobe
Oxygen
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Toxic forms of oxygen
normal ground state oxygentoxic forms of oxygen:
1. Singlet oxygen: O2 boosted to higher-energy state photochemically or
biochemically; extremelyreactive; caroteniods (pigments)convert to non-toxic forms
Oxygen
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Toxic forms of oxygen
other toxic forms are by-products of reductionof O2 H2O in respiration:
2. Superoxide anion (O2-): O2 + e- O2- ;very toxic because very
unstable, steal electrons from othermolecules, these in turn steal
electrons, etc.; superoxidedismutase (SOD) neutralises; aerobes,
facultative anaerobes, aerotolerantanaerobes produce SOD [ O2- + O2-
H2O2 + O2 ]
Oxygen
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Toxic forms of oxygen
3. Hydrogen peroxide: can damage cellcomponents, not as toxic as
superoxide or hydroxyl radical; catalase
[ 2H2O2 2H2O + O2 ] orperoxidase [ H2O2 + 2H+ 2H2O ]neutralise hydrogen peroxide
4. Hydroxyl radical: most reactive, instantlyoxidises any organic substance in cell;produced from hydrogen peroxide
[ H2O2 + e- + H+ H2O + OH ]
Oxygen
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Toxic forms of oxygen
O2- O2 + H2O2SOD
H2O
H2O + O2
Peroxidase
Catalase
Superoxide
Oxygen
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Toxic forms of oxygen
obligate anaerobes are extremely sensitive to O2obligate anaerobes produce neither SOD or catalase leads to accumulation of superoxide anions in
cytoplasm
aerotolerant anaerobes produce SOD or equivalentmicroaerophiles produce superoxide anions and
H2O2 in [lethal] in O2-rich conditions
Oxygen
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Toxic forms of oxygen
_________________________________________________________
Group SOD Catalase Peroxidase_________________________________________________________
Obligate aerobesand most facultative + + -anaerobes (e.g. Enterics)
Most aerotolerant anaerobes + - +(e.g. Streptococci)
Obligate anaerobes - - -(e.g. Clostridia)
_________________________________________________________-
Oxygen
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Toxic forms of oxygen
laboratory culture of obligate anaerobes:reducing media - contains ingredients
(sodium thioglycollate) that combine withdissolved O2 and deplete from media, usedin tubes, heated before use to drive offabsorbed O
2
culture grown on Petri dish to observeindividual colonies requires specialised
techniques
Oxygen
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Toxic forms of oxygen
laboratory culture of obligate anaerobes:anaerobic jars - O2 removed by adding H2O
to packet of sodium bicarbonate andsodium borohydrate; H2 and CO2 produced;
palladium catalyst in jar combines O2 with H2 H
2
O; O2
quickly disappears; CO2
helpsgrowth of anaerobes
Oxygen
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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) fittedto wall of chamber; hands inserted intogloves allows manipulation inside chamber.
Oxygen
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Anaerobic jar Anaerobic chamber
Oxygen
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Capnophiles
aerobes that grow better at higher [CO2] thanpresent in air
candle jar: lighted candle placed in sealed jar withcultures; candles stops burning when [O2] levelsfall; [CO2] elevated
CO2 incubators: electronic control of [CO2]commercial packets: contain CO2 generator; tube
crushed, chemicals mixed, reaction produces CO2to 10%; O
2reduced to 5%
Carbon
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one of the most important requirements for microbialgrowth (along with water)
structural backbone of living matterneeded for all organic compoundshalf of dry weight of cell is carboncarbon obtained from energy source -
carbohydrate, protein, lipids(chemoorganotrophs), organic compounds(photoheterotrophs), or from CO2(chemolithotrophs, photoautotrophs)
Other elements - N, S and P
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needed for synthesis of cellular materialproteins require N and SDNA, RNA, ATP require N and PN = 12% dry weightP and S = 4% dry weightN obtained from amino group of amino acids;proteins decomposed; amino acids incorporated
into new proteins and other compounds
Other elements - N, S and P
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other bacteria obtain N from NH4+ in cellularmaterial or nitrates (NO3-)
nitrogen fixers: use gaseous N2; importantprocess (nitrogen fixation); free-living
(photosynthetic cyanobacteria) orsymbiotic (Rhizobiumand legumes),nitrogen used by bacterium and plant
sulphur used in S-containing amino acids andvitamins (thiamine, biotin); sulphate
(SO42-), H2S and amino acids are sources of
sulphur
Other elements - N, S and P
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phosphorus essential for nucleic acids andphospholipids (cytoplasmic membranes)
high energy bonds of ATPphosphate (PO43-) important source of P
Other elements
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other elements required by microorganisms (co-factors for enzymes)K: required by protein synthesis enzymes
Mg: stabilises ribosomes, required foractivity of many enzymes
Ca: stabilises cell wall, heat stability ofendospores
Na: requirement depends of habitat(seawater vs freshwater)
Fe: key component of cytochromes(electron transport), siderophores areiron-binding agents that transport Fe
into cell
Trace elements
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required in very small amounts (micronutrients)metals (e.g. Mn, Mo, Ni, Zn)structural role in enzymesnaturally present in water, even distilled water,
and other media components, therefore notnormally added to laboratory media
Organic growth factors
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essential organic compounds unable to besynthesised (some vitamins in humans)
directly obtained from environmente.g. vitamins (co-enzymes), amino acids,
purines, pyrimidines
most bacteria can make all vitamins, somecannot; those unable to be made are organic growth factors
Culture media
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nutrient material prepared for growth ofmicroorganisms in the laboratory
some microorganisns cannot be grown onsynthetic media; need living host
Mycobacterium lepraegrown inarmadillos
obligate intracellular bacteria[rickettsias, chlamydias] and virusesreproduce only in living cells
Culture media
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criteria met by culture mediumcorrect nutrients for particular
microorganismsufficient moistureproperly adjusted pHsufficient level of O2 (sometimes none!)sterilecorrect incubation temperature
Culture media
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growth on solid media requires the addition ofagar (complex carbohydrate derived frommarine algae)
agar acts as a solidifying agentuseful properties of agar
few microbes can degrade liquifies at 100C, gels at 40C
Culture media
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Chemically defined media:
exact chemical composition is knownprecise amounts of purified chemicals added to
dH2O
Complex undefined (complex) media:
use digests of proteins (peptones), casein (milkprotein), beef, soybeans, yeast cells -
dissolved in dH2Ohighly nutritious but chemically undefinednutrient broth (liquid) or agar (solid)basal or enriched (fastidious organisms)
Culture media
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selectiveand differentialmedia, used in clinicaland public health microbiology
selective media:suppress the growth of unwanted
bacteriaencourage growth of desired onese.g. Bismuth sulphite agar
- selective for Salmonella- bismuth sulphite inhibits Gram
positive bacteria and most other
Gram negative bacteria
Culture media
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differential mediaallow desired microorganisms to bedistinguished 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
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-haemolysis -haemolysis
-haemolysis
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Culture media
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enrichment cultureused to when desired bacteria present in small
numbers, other bacteria in larger numbers
designed to increase very small numbers ofdesired organisms to detectable levels
often used for soil or faecal samplesmedium and incubation conditions selective
for desired organisms, counter-selective forothers
Pure cultures
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infectious materials, environmental, food samplescontain many different microorganisms
plating on solid medium visible colonies
one cell or spore one colony
distinctive colony morphologyallows identificationexperimentation and testing requires pure cultures;
derived from single colony, clones
Pure cultures
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streak plate methodobjective: to separate cells in inoculuminto
individual cells individual colonies
if desired microorganism is not present inlarge numbers, selective enrichmentoccurs prior to isolation by streak plate
method
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Pure cultures
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streak plate method
?
Growth of microorganisms
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growth = increase in number of cells = increase inmicrobial mass
increase in size of individual cellis insignificantcell 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 cellspinched off, cells separate
Growth of microorganisms
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Onegeneration
Growth of microorganisms
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growth rate = cell number (cell mass) / unittime
generation time = time required for cell todivide and population to double (doublingtime)
generation times:1-3 hours (bacteria)extremes = 10 minutes several hrs
daysE. coli= 20-30 minutes
Growth of microorganisms
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_____________________________________
Generation number Number of cellsArithmetic Logarithmic (log10)
_____________________________________
0 1 (= 20) 01 2 (= 21) 0.3012 4 (= 22) 0.6023 8 (= 23) 0.9034 16 (= 24) 1.204
5 32 (= 25) 1.50510 1,024 (= 210) 3.0115 32,768 (= 215) 4.51520 1,048,576 (= 220) 6.021
_____________________________________
Growth of microorganisms
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0 5 10 15 200
200000
400000
600000
800000
1000000
1200000
0
1
2
3
4
5
6
7
Arithmetic
Logarithmic
Generations
Arithmeticnu
mberofcells
L
ogarithmic
(log10)number
of
cells
Growth of microorganisms
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exponential growth: population increase wherecell number doubles per unit time
calculating generation times21 22, 22 23, 23 24 ...
N = N02n
N = final cell numberN0 = initial cell numbern = number of generations
Growth of microorganisms
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N = N02n
n = log N - log N0log 2
= log N - log N00.301
= 3.3 (logN - logN0)
Growth of microorganisms
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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 minutes14.5
Growth of microorganisms
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Four phases of bacterial growth (in liquid culture)
Lag phaseperiod of little or no cell division (1 hour -
several days)cells do not immediately reproducenot 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 topoorer one; damage to cells before culture
Growth of microorganisms
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Exponential (Log) phasehealthiest state of cellsone cell becomes tworates of exponential growth vary between
microbes (= slope on graph); influencedby growth conditions (temperature,
nutrients)
Stationary Phaseexponential growth cannot occur
indefinitelyessential nutrients used upwaste products build up
Growth of microorganisms
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no net increase or decrease in cell numbercryptic growth (cells continue to carry out
some metabolic activities)
Death Phaserate of cell death increasesexponential rate of decline, usually slower
than exponential growthtiny fraction of cells remain, or population
dies completely
can take a few days, some microbes canretain some surviving cells indefinitely
Growth of microorganisms
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Phases relate to populations of cells, not individual cells
Measurement of growth
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Population growth = changes in cell numbers orweight of cell mass
Methods for estimating cell numbers or massdirect methods
direct microscopic countmost probable numberviable (plate, colony) counts
indirect methodsturbidimetric measurementmetabolic activitydry weight
Direct microscopic count
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Breed count method (used to count bacteria inmilk):
measured volume (0.01 ml) of bacteria suspensionplaced in defined area (1 cm2) of slide
stain added to visualise cellsarea of viewing field determinednumber of bacteria counted (average of several
fields)
number of bacteria in suspension determined
Direct microscopic count
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area of viewing field determined by using stagemicrometer (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 = 1.5x10
5
cells/cm2
Original sample was 0.01 ml; therefore, X 100 to convertto ml, i.e. 1.5 x 105 x 100 = 1.5 x 107 cells/ml
Direct microscopic count
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Stage micrometer:
each division =0.01 mm
Direct microscopic count
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Haemocytometer (Petroff-Hauser cell counter)
Direct microscopic count
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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
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Limitations:cannot distinguish dead cells from live cellssmall cells difficult to see imprecisestaining to visualise cells (phase contrast if
unstained)
not suitable for low cell numbers (
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statistical estimating techniquepremise: more bacteria in a sample, more
dilution required to reduce density to pointwhere no cells left to grow in a series of
tubes
MPN tables consulted, used to determine thenumbers likely to give observed result
(refer to prac notes)
Viable counts
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most frequently used methodmeasures number of viable cellsassumes 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
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Two methods:spread plate: volume (0.1 ml) spread oversurfaceof 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 sterilePetri dish, molten agar added, mixed andallowed to set; colonies grow on surfaceandsubsurface; larger volumes can be used,organism must be able to withstand 45C
(molten agar)
Viable counts
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Spread plate
Pour plate
Viable counts
l d l
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Serial dilutions:for both methods, number of colonies cannot be too
large (overcrowding: not all cells colonies, fusionof colonies counting errors) or too small(statistical significance of counting)
only plates with 30-300 colonies are countedappropriate numbers obtained by serial dilutionseveral 10-fold dilutions of sample (sometimes
100-fold)
Viable counts
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10-1 10-2 10-3 10-4 10-5 10-6
Indirect methods
T bidi i f ll b
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Turbidimetric measurement of cell numberbacterial suspension looks cloudy (turbid), light
scattered: more cells, more turbid, more lightscattered
turbidity measured by spectrophotometer: passlight through suspension, measure unscatteredemergent light
absorbance or optical density (OD) = log10 (1/T)(T = percentage of transmission)OD proportional to cell number
Indirect methods
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1000
1000
Blank
Bacterialsuspension
Lightsource
Spectrophotometer
Indirect methods
M f b li i i f l i
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Measurement of metabolic activity of populationassumes that amount of a metabolic product
(acid, CO2) is proportional to cell number
Dry weightuseful for filamentous organisms (moulds)
where discrete colonies not formed
i d f h di