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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

Rate of things viaspectrophotometry

Aph 162, Winter 2009

Week 2

overview
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
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

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
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
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
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
Calibration – measuring background

Always need to measure “blank” - just medium.

The spectrophotometer subtracts this measurement from the actual measurement

a standard curve
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
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
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

growth phases
Growth phases
  • Lag phase
    • Occurs upon inoculation
    • Duration depends on history of inoculum (exponential/stationary/damaged/type of medium)
growth phases17
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
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
bacterial growth curves two carbon source catabolite repression
Bacterial growth curves –two carbon source: catabolite repression

Catabolism: biochemical reaction leading to production of usable energy

how does it work
How does it work?

CAP activator (constitutive)

LacI repressor

cAMP

Allolactose

CAP = catabolite activator protein

glucose

lactose

high glucose catabolite repression
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.

low glucose lactose switch
Low glucose: Lactose switch

OFF

High

glucose

Lactose:

High = ON

Low

glucose

Low = OFF

when will the diauxic shift occur
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
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
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?