There are six stations for you and your lab partner to visit on your lab tour today. Some will be guided tours with a TA or faculty there to help you and others are self-guided, leaving you and your partner to try things on your own. Your visit to each station will last 10-15 minutes. It doesn’t matter which station you visit first but you must visit them all before you leave today. Your lab practical next time will assess your mastery of each station.
Introduction to pipetting
Someone will show you how to use your pipetmen and then you will use them to dilute a blue dye (0.01% Xylene Cyanol).
- If you have never used pipetmen then you should practice by pipeting 800, 80 and 8 ul of the 0.01% XC stock into eppendorf tubes. XC is not hazardous but it will stain your clothes. Pipet each volume three times and visually inspect how well the volumes match.
- Using your P20, measure 10, 15 and 20 ul of the 0.01% XC stock solution into the bottom of three cuvettes. Using your P1000, add water to bring the final volume to 1 ml (=1000 ul).
- Using your P200, measure 20, 50 and 100 ul of the 0.01% XC stock solution into the bottom of three more cuvettes. Using your P1000, add water to bring the final volume to 1 ml.
- Using your P1000, measure 100, 200, and 400 ul of 0.01% XC solution into the bottom of three more cuvettes. Add water to bring the final volume to 1 ml.
- With a gloved hand or with a piece of parafilm over the lip of the cuvette, invert each cuvette several times to thoroughly mix the contents.
- Visually compare your dilutions to the reference ones. If time permits, you will read the absorbance of your dilutions in the spectrophotometer so do not throw them away.
Introduction to our microscopes
Much of biology examines natural components that are too small to see. Imaging technology took a gigantic step forward in the 1680s when Anton van Leeuwenhoek ground a microscope lens sufficiently fine to see a living cell (a bacteria he had scraped from his teeth!). His microscope had one lens and the image he saw was approximately 250 times its natural size (250X magnification). Compound microscopes, like the ones we have in lab, use a second lens to magnify the image from the first and can increase the total magnification up to 1000X. One of our microscopes is also attached to a beam splitter that allows excitation light to be separated from emitted light. This allows us to perform fluorescence microscopy.
No matter how fine its lens, a light microscope cannot distinguish objects closer than 200 nm. The resolution of light microscopes is limited by both the wavelength of white light (300-700 nm) and the scattering of light by the object it strikes. For better resolution, great lenses must be combined with shorter wavelengths, such as those followed by electrons or lasers, and better ways of focusing the beam such as forcing it to travel through a vacuum or an oil. Linking the microscope to a computer with digital image processing can also enhance its images. The sample itself can also be stained or fluorescently tagged to improve detection of its features.
Today you will be shown how to use each of the microscopes in the main lab and you will use them to compare three cell types. You will be asked to focus a sample during the lab practical next week.