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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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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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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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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)


(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


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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)


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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-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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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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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


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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Bi s nth sis f min ids

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Amination:

-Ketoglutarate + NH3 GlutamateGlutamate

dehydrogenase

Glutamate + NH3 GlutamineGlutamine

synthetase

NH2

NH2 NH2 NH2


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Transamination:

Glutamate + OxalacetateTransaminase

-Ketoglutarate + Aspartate

NH2

NH2

Biosynthesis of fatty acids

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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


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Glucose

Glyceraldehyde-3-phosphate

Acetyl CoA

CAC

G

lycolysis

Dihydroxyacetonephosphate

GlycerolPyruvate

Fatty acids

Lipids


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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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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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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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O2- O2 + H2O2SOD

H2O

H2O + O2

Peroxidase

Catalase

Superoxide

Oxygen

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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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_________________________________________________________

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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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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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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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


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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


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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


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Onegeneration


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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


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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

_____________________________________


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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


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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


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N = N02n

n = log N - log N0log 2

= log N - log N00.301

= 3.3 (logN - logN0)


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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


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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


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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


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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


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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


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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


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Stage micrometer:

each division =0.01 mm


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Haemocytometer (Petroff-Hauser cell counter)


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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


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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

topic 2_2012

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