During the first skills test, some of you were able to answer the question relating to glycerol, but most were not. There was a statement that suggested you try pipetting the glycerol solution at the front of the lab. However, many of you attempted to answer the question "theoretically". I cannot stress the importance of experiments and this is one simple example of why it was necessary to at least attempt to pipette the glycerol.
Like a number of compounds, glycerol is viscous, it is more like motor oil, or a thick shampoo than water and it has a density (see here for a refresher) that is greater than water (which is 1, and is why 1ml of water weighs 1g). It is sometimes necessary to think laterally when preparing solutions and in this case, weighing the liquid is one way of ensuring that an accurate volume of the stock glycerol liquid is added to water to make a final dilution. And it may be necessary to make a larger volume of the solution than you need depending on the availability of precision balances.
I noticed today that many of you assumed that the dilutions needed to obtain a reliable standard curve for Crystal Violet determinations, were far too great. In the experiments last week we used a much more concentrated stock of dye, hence the large dilutions necessary to obtain absorbances of less than 1. This week however, the stock dye was diluted in advance and so smaller dilutions were required. When carrying out similar experiments, it is good practice to make a dilution that will give you a reasonable level of colour: not too dark and not too weak. Measure the absorbance and use this measurement to inform the range of dilutions needed to obtain a standard curve with absorbance values between 0.1 and 0.8.
Key words: Absorbance, standard curve, cuvettes, spectroscopy
Three weeks into the new term and we have been going through the basic skills required to make accurate dilutions using automatic pipettes. Using a range of microbiological dyes, UTC students from Y10-13 are coming to terms with the "reliability" of these pipettes for delivering microlitre volumes in order to produce high quality standard curves using the Beer Lambert Law. The main aim of these experiments is to develop ownership of your experimental results and to keep practising until you get a result that is "trustworthy" and one that is unequivocal. At this stage on your journey, the most important thing is to ensure you understand the significance of each step in a protocol and that you appreciate when to use (in this case) a P1000, a P20 etc in preparing dilutions or reaction mixtures for PCR etc.
My one comment is that pipetting 1 microlitre quantities and diluting with 999 microlitres is likely to be a less accurate way of making a 1000-fold dilution than making two or more dilution steps. For example, mix 100 microlitres of your concentrate with 900 microlitres followed by a second dilution of say 10 microlitres of the first dilution with 990 microlitres of water. Also, the P5/P10 pipettes are mainly used when you have no choice i.e. when you are pipetting valuable, sterile or possibly hazardous samples. Finally, when you see data points that fall off an expected line, as top left (in the current classes, the absorbance versus concentration plot), simply repeat the measurement together with one or two of the samples that fall on your expected line. It is good practice to plot all measurements and not just the mean value.
Key Words: Pipette, Gilson, standard curve, dilution, volumetric.
