Spectrophotometry Biophotonics • Biophotonics – study of the interaction of biological materials with light and other forms of radiant energy. • Radiation - energy that comes from a source and can travel through material or space 1 Biophotonics • Light interacts with organic compounds – Reflection – Absorption – Transmission – Scattering 2 Spectrophotometry • Quantitative study of the electromagnetic spectrum. • Spectrophotometer – measures the absorption of light by a substance. • We will use a UV/Visible spectrophotometer to measure the concentration of bacteria and DNA 3 Spectrophotometry • Wavelength of absorbance (An)– wavelength which the spectrophotometer is set to absorb. – Example: bacteria absorb light at a wavelength of 600nm (A600) • Optical Density (OD) – Amount of absorbed light per thickness of the sample. Measuring Bacterial Growth • Bacterial growth occurs in four stages. – lag phase • bacteria is added to the media – log or exponential phase • bacteria are reproducing at their maximum rate – saturation or stationary phase • growth rate of the bacteria slows during this phase due to the depletion of nutrients and the accumulation of waste products – death phase • bacteria begin to run out of nutrients and die 4 Measuring Bacterial Growth • Techniques – Hemocytometer • Used to count the number of cells. • Drawback – total number of cells not the number of cells which are alive. – Serial dilution – Spectrophotometry • The amount of light absorbed by the bacterial culture is measured • A wavelength of 600nm (A600) is used for measuring bacterial concentration • Quick and easy! 5 Measuring Bacterial Growth • Harvest a bacterial culture during the early log phase of cell growth. – OD of .5-.7 – Cells are at peak growth rate. DNA Purity • A purity ratio is often used to determine the relative purity of a sample of DNA or RNA • DNA and RNA both absorb at a wavelength of 260nm • Contaminants in the sample, such as proteins, absorb at a wavelength of 280nm • A260/A280 – provides information on the relative purity of the DNA sample – A reading of 1.8 indicates a pure DNA sample – A reading of 2.0 indicates a pure RNA sample. 6 DNA Concentration • Concentration = A260 x Conversion Factor x Dilution Factor • Conversion factor DNA Purity and Concentration • Important! Use a quartz cuvette when measuring RNA or DNA concentrations! • Glass and plastic cuvettes absorb UV wavelengths. • BE EXTREMELY CAREFUL WHEN HANDLING THE QUARTZ CUVETTE! AVOID FINGERPRINTS ON THE SURFACE! DO NOT DROP THE CUVETTE! 7 DNA Purity and Concentration General Guidelines • Be careful not to touch cuvettes on the smooth surface. Fingerprints will distort your readings. • Choose an assay • Blank the spectrophotometer using fresh media. • Take a reading. 8.