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Rate of things via spectrophotometry. Aph 162, Winter 2009 Week 2. Overview. Spectrophotometry The Beer-Lambert law Some weird units: OD 600 and cfu ’s Calibration: a standard curve (OD 600 vs. cfu) Bacterial growth curves Growth on a single carbon source

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Rate of things via spectrophotometry l.jpg

Rate of things viaspectrophotometry

Aph 162, Winter 2009

Week 2


Overview l.jpg
Overview

  • Spectrophotometry

    • The Beer-Lambert law

    • Some weird units: OD600 and cfu’s

    • Calibration: a standard curve (OD600 vs. cfu)

  • Bacterial growth curves

    • Growth on a single carbon source

    • Growth on a two carbon sources

      (diauxic growth/catabolite repression)

  • Experiments for today


Spectrophotometry the beer lambert law l.jpg
Spectrophotometry: The Beer-Lambert law

  • Relates concentration to the optical measurement of ‘absorbance’

    • Example: E. coli concentration

  • Combined with spectrophotometry can be used to distinguish and compare different molecules in solution

    • Example: Chlorophyll spectrum




The beer lambert law l.jpg

z

σ

The Beer-Lambert law

  • I0 = incident light (W/cm^2)

  • c = Number density of absorbers (e.g. cells)

  • σ(λ)= particle cross section (cm^2)

  • l = width of cuvette(usually 1cm)


The beer lambert law7 l.jpg
The Beer-Lambert law

z

σ

  • I0 = incident light (W/cm^2)

  • c = Number density of absorbers (e.g. cells)

  • σ(λ)= particle cross section (cm^2)

  • l = width of cuvette (usually 1cm)

  • For dilute samples:

    dIz/Iz =-σ·c·dz

    I1 (λ) = I0e-σ(λ)·c·l= I010-ε(λ)·c·l


The beer lambert law8 l.jpg
The Beer-Lambert law

z

σ

  • I0 = incident light (W/cm^2)

  • c = Number density of absorbers (e.g. cells)

  • σ(λ)= particle cross section (cm^2)

  • l = width of cuvette (usually 1cm)

  • For dilute samples:

    dIz/Iz =-σ·c·dz

    I1 (λ) = I0e-σ(λ)·c·l= I010-ε(λ)·c·l

  • Absorbance=A(λ)= -log(I1/I0)=ε·c·l


The beer lambert law9 l.jpg
The Beer-Lambert law

z

σ

  • I0 = incident light (W/cm^2)

  • c = Number density of absorbers (e.g. cells)

  • σ(λ)= particle cross section (cm^2)

  • l = width of cuvette (usually 1cm)

  • For dilute samples:

    dIz/Iz =-σ·c·dz

    I1 (λ) = I0e-σ(λ)·c·l= I010-ε(λ)·c·l

  • Absorbance=A(λ)= -log(I1/I0)=ε·c·l

  • ODλ=600=A/l = ε(λ=600nm)·c ~ c

  • Units of OD: per unit length



Calibration measuring background l.jpg
Calibration – measuring background

Always need to measure “blank” - just medium.

The spectrophotometer subtracts this measurement from the actual measurement


A standard curve l.jpg
A standard curve

  • OD600 doesn’t give absolute cell concentration

  • OD600 is cell dependent

  • Need to independently measure cell concentration so that the two can be related. This is called a standard curve.


A standard curve cont l.jpg
A standard curve (cont.)

  • Measure absolute cell concentration by dilution and plating.

  • Plating measures cfus = colony forming units

  • Standard curve = plot OD600 vs. cfu


How to do it in the lab l.jpg
How to do it in the lab

Plate every 30min

Try DX10 and D/10 as well

Next day:

http://micro.fhw.oka-pu.ac.jp/lecture/exp/images/cfu-7.jpg


Bacterial growth curves single carbon source l.jpg
Bacterial growth curves – single carbon source


Growth phases l.jpg
Growth phases

  • Lag phase

    • Occurs upon inoculation

    • Duration depends on history of inoculum (exponential/stationary/damaged/type of medium)


Growth phases17 l.jpg
Growth phases

  • Exponential phase

    • Healthy cells

    • Cell number increases exponentially with a well defined doubling time

    • Reproducible physiological state

    • OD600 ~ 0.1

    • Doubling times can be 20mim, hours, weeks and even months depending on the organism and growth medium


Growth phases18 l.jpg
Growth phases

  • Stationary phase

    • Population reaches steady state because

      • An essential nutrient becomes limiting

      • A waste product generated by the culture inhibits further growth

    • Physiological state of cell completely changes: cells are in stress


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Bacterial growth curves –two carbon source: catabolite repression

Catabolism: biochemical reaction leading to production of usable energy


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How does it work?

CAP activator (constitutive)

LacI repressor

cAMP

Allolactose

CAP = catabolite activator protein

glucose

lactose


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High glucose: Catabolite repression

OFF

High

glucose

When glucose is present → no activator → this operon as well as operons for other sugars are shut off.


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Low glucose: Lactose switch

OFF

High

glucose

Lactose:

High = ON

Low

glucose

Low = OFF


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When will the diauxic shift occur?

Experimental setup:

  • 1L of glucose at 0.1g/L

  • Inoculums at t=0 is 10mL of saturated E. coli culture

    (@ OD600 = 1.5)

  • Rich medium (with casamino acids)

  • Doubling time: 20 min

  • Aerobic growth


Experiments for today l.jpg
Experiments for today

  • Choose a growth medium

    • Glucose+Lactose/Matlose/Sorbitol (1:1 ratio, 0.1 g/L)

  • Measure OD600 every 5-10min (esp. near shift)

    • Don’t forget to blank before each measurement!

    • Minimize time incubator is open

  • Shift should occur at OD~0.25

  • Every ~30 min plate cells

    • Remember: OD600=1 ↔ 109 cells/mL

  • Note absolute time


Homework l.jpg
Homework

  • Plot growth curve on a log scale

    • Identify all growth phases

    • Analyze your results in light of our discussion on catabolite repression

  • Extract doubling times by linear regression

    • Do your values make sense?

  • Plot standard curve (OD600 vs. cell count)

    • Is it linear? Are there errors? Why?


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