Rate of things via spectrophotometry

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# Rate of things via spectrophotometry - PowerPoint PPT Presentation

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 viaspectrophotometry

Aph 162, Winter 2009

Week 2

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
• 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

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 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 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 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

Always need to measure “blank” - just medium.

The spectrophotometer subtracts this measurement from the actual measurement

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.)
• 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

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
• Lag phase
• Occurs upon inoculation
• Duration depends on history of inoculum (exponential/stationary/damaged/type of medium)
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 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

Catabolism: biochemical reaction leading to production of usable energy

How does it work?

CAP activator (constitutive)

LacI repressor

cAMP

Allolactose

CAP = catabolite activator protein

glucose

lactose

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

OFF

High

glucose

Lactose:

High = ON

Low

glucose

Low = OFF

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
• 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
• 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?