SQA Advanced Higher Biology

Hazard and risk in the laboratory

A hazard is anything that could cause harm, and risk is the likelihood that harm will actually arise from exposure to that hazard. A bottle of concentrated acid is a hazard whether or not anyone touches it; the risk depends on how much is used, how it is handled and who is nearby. The distinction matters because a risk assessment does not try to remove hazards from a laboratory, which is impossible in biology. It identifies each hazard, judges how likely and how serious the harm would be, and then decides on control measures that reduce the risk.

The hazards in a biology laboratory fall into a few groups.

Control measures are matched to the hazard. They include handling techniques that keep a substance away from skin and eyes, protective clothing and equipment such as gloves, lab coats and eye protection, working in a fume cupboard for volatile chemicals, balancing a centrifuge so that it cannot fly apart, and aseptic technique for organisms, which keeps cultures from escaping into the room as well as keeping contaminants out of the culture. A sound risk assessment names the hazard, states who could be harmed and how, gives the control measure, and says what to do if it fails, such as flushing a splash with water or reporting a spill.

Linear and log dilution series

Solutions in the laboratory are very often made by diluting a stock solution, and two kinds of series are used.

In a linear dilution series the concentrations differ by an equal interval. A series of 0.1, 0.2, 0.3, 0.4 and 0.5 mol dm⁻³ goes up in steps of 0.1. This is useful when the response being measured changes steadily with concentration over a modest range, and when a graph with evenly spaced points is wanted.

In a log dilution series each solution differs from the last by a constant proportion, usually a factor of ten, so the dilutions are $10^{-1}$, $10^{-2}$, $10^{-3}$ and so on. Each step takes one volume of the previous solution and adds nine volumes of diluent. A log series covers a huge range of concentrations in a few tubes, which suits situations where a quantity such as the number of bacteria in a culture could lie anywhere across several orders of magnitude. The dilution factor is the same at every step, and the overall dilution after several steps is the product of the individual factors.

Worked example

A student wants a series from a 1.0 mol dm⁻³ stock, diluted by a factor of ten each time, and needs 10 cm³ of each solution.

For each step, 1 cm³ of the previous solution is added to 9 cm³ of diluent, which gives 10 cm³ at one tenth the concentration.

For a linear series of 0.2, 0.4, 0.6 and 0.8 mol dm⁻³ made from the same stock, the volume of stock required in a final 10 cm³ is given by

so for 0.4 mol dm⁻³ the volume is $\frac{0.4 \times 10}{1.0} = 4\ \text{cm}^3$ of stock made up to 10 cm³ with 6 cm³ of diluent.

The accuracy of a dilution series depends on the measuring equipment. Measuring cylinders are suitable for larger volumes where small errors matter little, pipettes and burettes deliver more precise volumes, autopipettes and syringes are used for the very small volumes needed in molecular work, and a fresh tip or pipette is used for each step so that concentrated solution is not carried into the next tube.

Standard curves

A standard curve is a graph made from measurements on solutions of known concentration, used to find the concentration of an unknown. The technique is used whenever a property that can be measured (absorbance, for example) changes with the amount of a substance that cannot easily be measured directly.

The steps are these.

1. Make a dilution series of the substance, for example a protein, at known concentrations.
2. Measure the property (such as absorbance in a colorimeter) for each of the known solutions, under exactly the same conditions as will be used for the unknown.
3. Plot the measured values on the vertical axis against the known concentrations on the horizontal axis and draw a line or curve of best fit through the points.
4. Measure the same property in the unknown sample, find that value on the vertical axis, read across to the curve and then down to the horizontal axis to give the concentration.

The unknown must fall within the range of the standards. Reading off a value beyond the highest or lowest standard means extending the line into a region where its shape is unknown, and the answer cannot be trusted. If the unknown is too concentrated it is diluted by a known factor and measured again, and the concentration read from the curve is multiplied by that factor. If the relationship is not a straight line, a curve is drawn rather than forcing a line through the points.

A blank, which contains everything except the substance being measured, is included so that the contribution of the solvent, reagents and container can be removed from every reading.