1. Rate of things via spectrophotometry Aph 162, Winter 2009 Week 2

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 –Growth on a two carbon sources (diauxic growth/catabolite repression) Experiments for today

3. 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

4. Spectrophotometer

6. z σ The Beer-Lambert law I 0 = 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)

7. z σ The Beer-Lambert law I 0 = 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: dI z /I z =-σ·c·dz I 1 (λ) = I 0 e -σ(λ)·c· l = I 0 10 -ε(λ)·c· l

8. z σ The Beer-Lambert law I 0 = 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: dI z /I z =-σ·c·dz I 1 (λ) = I 0 e -σ(λ)·c· l = I 0 10 -ε(λ)·c· l Absorbance=A(λ)= -log(I 1 /I 0 )=ε·c· l

9. z σ The Beer-Lambert law I 0 = 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: dI z /I z =-σ·c·dz I 1 (λ) = I 0 e -σ(λ)·c· l = I 0 10 -ε(λ)·c· l Absorbance=A(λ)= -log(I 1 /I 0 )=ε·c· l OD λ=600 =A/ l = ε(λ=600nm)·c ~ c Units of OD: per unit length

10. When the law is applicable

11. Calibration – measuring background Always need to measure “blank” - just medium. The spectrophotometer subtracts this measurement from the actual measurement

12. A standard curve OD 600 doesn’t give absolute cell concentration OD 600 is cell dependent Need to independently measure cell concentration so that the two can be related. This is called a standard curve.

13. A standard curve (cont.) Measure absolute cell concentration by dilution and plating. Plating measures cfus = colony forming units Standard curve = plot OD 600 vs. cfu

14. How to do it in the lab http://micro.fhw.oka-pu.ac.jp/lecture/exp/images/cfu-7.jpg Plate every 30min Next day: Try DX10 and D/10 as well

15. What you need to know OD 600 =1 ↔ 10 9 cells/mL Calculating the dilution factor D: 0.1mL X OD 600 X 10 9 cells/mL / D = 100 cells Try DX10 and D/10 as well

16. Bacterial growth curves – single carbon source

17. Growth phases Lag phase –Occurs upon inoculation –Duration depends on history of inoculum (exponential/stationary/damaged/type of medium)

18. Growth phases Exponential phase –Healthy cells –Cell number increases exponentially with a well defined doubling time –Reproducible physiological state –OD 600 ~ 0.1 –Doubling times can be 20mim, hours, weeks and even months depending on the organism and growth medium

19. 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

20. Bacterial growth curves –two carbon source: catabolite repression Catabolism: biochemical reaction leading to production of usable energy

21. How does it work? CAP activator (constitutive) cAMP glucose LacI repressor Allolactose lactose CAP = catabolite activator protein

22. High glucose: Catabolite repression High glucose OFF When glucose is present → no activator → this operon as well as operons for other sugars are shut off.

23. Low glucose: Lactose switch High glucose OFF Low glucose High = ON Lactose: Low = OFF

24. 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 (@ OD 600 = 1.5) Rich medium (with casamino acids) Doubling time: 20 min Aerobic growth

25. Experiments for today Choose a growth medium –Glucose+Lactose/Matlose/Sorbitol (1:1 ratio, 0.1 g/L) Measure OD 600 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: OD 600 =1 ↔ 10 9 cells/mL Note absolute time

26. 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 (OD 600 vs. cell count) –Is it linear? Are there errors? Why?