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What is Microbiology?What is Microbiology?

Unicellular Organisms

• Bacteria

• Viruses

• Fungi

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The Prokaryotic Bacterial CellThe Prokaryotic Bacterial Cell

• bacteria are unicellular

• multiply by binary fission

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Bacterial CellsBacterial CellsSize

– 0.20 to 2.0 um in diameter – 2 to 8 um in length.

Shape – spherical (coccus) – rod (bacillus) – spiral – pleomorphic

Arrangement

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Species differentiationSpecies differentiation

morphology chemical composition nutritional requirements biochemical activities source of energy DNA sequence

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Structures External to Cell WallStructures External to Cell Wall

• Capsule

• Flagella

• Fimbriae (pili)

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Bacterial cell wallBacterial cell wall

• Semi-rigid structure gives cell its shape• Protects cell from rupture due to osmotic stress• Composed of peptidoglycan (PG): repeating units

of n-acetylglucosamine (NAG) & n-acetylmuramic acid (NAM) linked by peptide bridges

• Peptide composition and thickness varies among species

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Gram positive/negative cell-wallsGram positive/negative cell-walls

• Gram-positive– Thick PG (10-100nm)– 90% of CW

– Single membrane

• Gram-negative– Thin PG (2nm)– 20% of CW

– Inner membrane and outer membrane

– Periplasmic space

– LPS

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Experiment 1: Gram StainDeveloped by Hans Christian Gram in Denmark (1844)

1. Bacterial suspension dried on glass slide

2. Crystal violet 1 minute. Wash off.

3. Iodine 1 minute. Wash off.

4. 95% alcohol 10 seconds. Wash off.

5. Safranin or basic fuschsin 30 seconds

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Gram StainGram StainCrystal violet + iodine = dye-I2 complexes in

cytoplasm (I2 stabilizes crystal violet)

• In G-, de-colorizer dissloves lipid layer, diffuses through cell-wall and decolorizes crystal violet. 2nd stain taken up.

• In G+, wall is too thick to allow uptake of de-colorizing agent and remains violet

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Gram StainGram Stain

Fix

Crystal violet

Iodine

De-colorize

(acetone-alcohol)

Safranin/fuschin

Gram-positive Gram-negative

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Gram-positive & Gram-negative bacteriaGram-positive & Gram-negative bacteria

• Gram-positive: – Mostly cocci (staphylococci, streptococci,

pneumococci, enterococci)– Some bacilli (bacillus, clostridia,

corynebacteria

• Gram-negative– Mostly bacilli (E.coli, H. pylori, Salmonella, V.

cholerae– Some cocci (Neisseria, Moraxella)

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Experiment 2 : Bacterial GrowthExperiment 2 : Bacterial Growth

Determination of bacterial viable count in liquid culture

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Bacterial growth

• Bacteria are unicellular

• Growth occurs by cell-division

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Cell DivisionCell Division

• Increase in Increase in CELL MASSCELL MASS• Increase in number of Increase in number of RIBOSOMESRIBOSOMES

• necessary cell structures, components, etc. necessary cell structures, components, etc.

• Duplication of Duplication of CHROMOSOMECHROMOSOME• DNA replicationDNA replication

• Synthesis of new Synthesis of new CELL WALLCELL WALL• PG: NAG & NAMPG: NAG & NAM

• Partitioning of Partitioning of TWO CHROMOSOMESTWO CHROMOSOMES• Formation of Formation of SEPTUMSEPTUM

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Bacterial growthBacterial growth

• Divided into 4 distinct stagesDivided into 4 distinct stages• LAGLAG

• LOGLOG

• STATIONARYSTATIONARY

• DEATHDEATH

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Bacterial growth kineticsBacterial growth kinetics

StationaryStationary

LogLog DeathDeath

LagLag

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Lag PhaseLag Phase

• Physiological adaptation to environmentPhysiological adaptation to environment

• Time varies Time varies

• recovery from “shock”recovery from “shock”

• different enzymes / proteins may be required for growthdifferent enzymes / proteins may be required for growth

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Log PhaseLog Phase

• binary fissionbinary fission

• optimal conditions for growthoptimal conditions for growth

• logarithmic or exponentiallogarithmic or exponential

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Exponential growth (log phase)

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Bacterial generation time

• Time it takes bacterial population to double in size (number) during log-phase growth

• Time it takes for population to double in size does not change with cell # (only true in log phase)

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Generation timeGeneration time

• 20 minutes to 20 hours depending on the bacterial species/strain and conditions during which log-phase growth is occurring

– Escherichia coliEscherichia coli• 20-60 minutes (20-60 minutes (in vitro)in vitro)• 5-10 hours (5-10 hours (in vivoin vivo))

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Stationary phaseStationary phase

• ““exhaustion of space”exhaustion of space”

• ““biological space”biological space”

• secondary metabolites – antibioticssecondary metabolites – antibiotics

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Stationary phaseStationary phase

• steady-state equilibriumsteady-state equilibrium• rate of cell growth = rate of cell deathrate of cell growth = rate of cell death

• loss of limiting nutrientsloss of limiting nutrients

• build-up of toxinsbuild-up of toxins

DJB/MBY376/Winter 2004/Bacterial GrowthDJB/MBY376/Winter 2004/Bacterial Growth

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Death phaseDeath phase

• ““die-off” occurs exponentiallydie-off” occurs exponentially

• viable cell population dies offviable cell population dies off

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Measurement of Bacterial GrowthMeasurement of Bacterial Growth

• Cell numbersCell numbers

• Cell massCell mass

• Metabolic rateMetabolic rate

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Cell numbersCell numbers

• Direct microscopic countDirect microscopic count

• Viable count (colony forming units)Viable count (colony forming units)

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Cell massCell mass

• Dry weight versus wet weightDry weight versus wet weight

• Volume of cells after centrifugationVolume of cells after centrifugation

• Measurement of total N or proteinMeasurement of total N or protein

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Cell mass / numbersCell mass / numbers

• TurbidityTurbidity

Metabolic rateMetabolic rate

• 002 2 uptake, C0 uptake, C022 and ATP production and ATP production

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Lab SafetyLab Safety

• Normal lab practice i.e. lab coats, no eating etc

• Handling of pathogens

• Aseptic technique