Practical skills Revision Notes

Experimental Setup for Investigating Photosynthesis

To investigate factors affecting the rate of photosynthesis, a common setup involves using an aquatic plant like pondweed (Elodea). The plant is submerged in a beaker of water (often with added sodium hydrogencarbonate to provide carbon dioxide) and illuminated by a light source. The oxygen gas produced during photosynthesis can be collected and measured. A method involves placing a funnel over the pondweed and an inverted measuring cylinder or boiling tube over the funnel, filled with water, to collect the gas bubbles. The volume of oxygen collected or the number of oxygen bubbles released per minute can be used as a measure of the rate of photosynthesis. Control variables, such as temperature, carbon dioxide concentration, and the age/size of the plant, must be kept constant to ensure a fair test.

Independent, Dependent, and Control Variables

When investigating photosynthesis, it's essential to correctly identify the variables. The independent variable is the factor you change to see its effect, for example, light intensity (by varying the distance of a lamp from the pondweed), temperature (using a water bath), or carbon dioxide concentration (by adding different amounts of sodium hydrogencarbonate). The dependent variable is what you measure, which in photosynthesis investigations is typically the rate of oxygen production (number of bubbles per minute or volume of gas collected per unit time). Control variables are factors kept constant to ensure the experiment is fair and valid, such as the type and amount of pondweed, the volume of water, the concentration of bicarbonate, and the duration of observation.

Measuring and Interpreting Results

The rate of photosynthesis can be measured by counting the number of oxygen bubbles released by the pondweed in a set time (e.g., one minute) or by measuring the volume of oxygen gas collected in a gas syringe or inverted measuring cylinder over a longer period. Repeating measurements for each independent variable setting allows for calculation of averages, reducing the impact of random errors, and helps to identify anomalous results. Results are usually plotted on a graph, with the independent variable on the x-axis and the rate of photosynthesis on the y-axis. This allows for visual identification of trends, such as how increasing light intensity or carbon dioxide concentration initially increases the rate of photosynthesis, until another factor becomes limiting.

Safety and Evaluation of Investigations

Safety precautions should always be observed in investigations. When using a lamp as a light source, ensure it's not too close to the water to prevent electrocution hazards, and be aware that the lamp can get hot. Hot water baths used for temperature control also pose a burn risk. After collecting results, it's important to evaluate the investigation. Consider potential sources of error, such as inconsistencies in bubble size when counting, variations in the pondweed's health or surface area, or inaccurate timing. Suggesting improvements, such as using a gas syringe for more accurate volume measurement, better temperature control, or repeating the experiment multiple times, helps to improve the reliability and validity of the experiment.

Ecological Sampling Techniques

Ecological sampling allows us to estimate the distribution and abundance of organisms in a habitat without counting every single one. Two primary methods are used: quadrats and transects. Quadrats are square frames of a known area used for stationary or slow-moving organisms. They must be placed randomly within the sampling area to ensure unbiased data. Random placement can be achieved by using random number generators to determine coordinates. Multiple samples increase the reliability of the results. Transects are used to investigate changes in organism distribution across an environmental gradient. A line transect records organisms touching a line at regular intervals, while a belt transect uses quadrats placed at regular intervals along the line to gather more detailed abundance data. Both methods allow for correlation between organism distribution and environmental factors.

Measurements and Variables in Sampling

When using quadrats, common measurements include frequency (how often a species appears in quadrats) and percentage cover (the proportion of a quadrat area covered by a species). For transects, data often shows how the number or percentage cover of organisms changes with distance along the gradient. It is vital to identify variables. The independent variable is the factor being changed (e.g., distance along a transect, light intensity) and the dependent variable is what is measured (e.g., number of daisy plants, percentage cover of moss). Control variables are factors kept constant to ensure a fair test, such as the size of the quadrat used or the exact measurement technique employed. Environmental factors like light intensity, soil pH, temperature, and moisture levels should also be measured along the transect to identify potential correlations. These measurements help to establish cause-and-effect relationships or strong correlations between environmental conditions and the species present or their abundance.

Methodology for Quadrat Sampling

To conduct a fair and reliable quadrat sampling investigation, follow these steps: 1. Choose an appropriate quadrat size based on the organisms being studied. 2. Mark out the sampling area. 3. Use random number generation to obtain coordinates for placing the quadrat. For example, if your area is 10m x 10m, generate two random numbers between 0 and 10 for the x and y coordinates. 4. Place the quadrat at the generated coordinates. 5. Count or estimate the coverage of the target organism(s) within the quadrat. 6. Repeat this process at least 10-20 times across the sampling area to obtain a representative sample. 7. Calculate the average number or percentage cover per quadrat. Safety: Be aware of uneven terrain, stinging plants, and protective clothing during fieldwork. Always work with an adult or in a group.

Methodology for Transect Sampling

To investigate changes along an environmental gradient using a transect: 1. Lay down a tape measure across the area where the environmental factor changes (e.g., from shaded to sunny or wet to dry). This forms your transect line. 2. At regular intervals along the transect (e.g., every 0.5 m or 1 m), place a quadrat. 3. Within each quadrat, record the number of target organisms or their percentage cover. 4. At each sampling point (where the quadrat is placed), measure the relevant environmental factor(s) such as light intensity (with a light meter), soil pH (with a pH meter or indicator), or soil moisture (with a moisture meter). 5. Repeat the transect in different areas if looking for overall trends, or take several parallel transects to increase reliability. Safety: Similar to quadrat sampling, consider terrain and plants. Handle environmental probes carefully.

Aseptic Techniques for Microbial Investigations

Aseptic techniques are crucial when working with microorganisms to prevent contamination of cultures by unwanted microbes and to prevent the spread of microorganisms to people or the environment. Key techniques include sterilising all equipment (e.g., inoculating loops, petri dishes, culture media) before use, typically using a Bunsen burner flame or an autoclave. Working near a lit Bunsen burner creates an updraft, drawing microbes away from the working area. Petri dishes should be opened minimally and quickly, and never fully inverted until sealed, to reduce airborne contamination. Wear appropriate PPE, such as lab coats and gloves, and disinfect the work surface before and after the experiment. Proper disposal of microbial cultures and contaminated materials, often by autoclaving, is also an essential aseptic practice.

Investigating Antimicrobial Effectiveness: Method

To investigate the effectiveness of antimicrobial agents: 1. Sterilise all equipment, including Petri dishes, agar, and inoculating loops. 2. Prepare an agar plate inoculated with a bacterial culture, spreading the bacteria evenly across the surface. 3. Using sterile forceps, place paper discs, each soaked in a different antimicrobial agent (or different concentrations), onto the surface of the inoculated agar. Include a control disc soaked in sterile water. 4. Seal the Petri dish with tape, but do not seal completely around the entire circumference to allow for aerobic respiration of the bacteria and prevent the growth of anaerobic pathogens. 5. Incubate the plate upside down at a suitable temperature (e.g., 25°C for school labs to prevent growth of human pathogens) for 24-48 hours. 6. Observe and measure the diameter of any clear zones (zones of inhibition) around the discs. The larger the zone, the more effective the antimicrobial agent.

Variables and Controls

In an experiment investigating antimicrobial effectiveness:

• Independent Variable: The type or concentration of the antimicrobial agent being tested. This is what you change.
• Dependent Variable: The diameter of the zone of inhibition around each disc. This is what you measure.
• Control Variables: Factors kept constant to ensure a fair test. These include: the species of bacteria used, the volume of bacterial culture inoculated, the size and material of the paper discs, the type of agar medium, the incubation temperature, and the incubation time. A control disc soaked in sterile water (no antimicrobial agent) is essential to show that bacterial growth would occur without the agent.

Data Analysis and Evaluation

After incubation, measure the diameter of the clear zone (zone of inhibition) around each antimicrobial disc. Take measurements across different diameters for each zone and calculate an average to improve accuracy. Repeat the entire experiment multiple times to ensure the reliability and reproducibility of the results. Calculate the mean diameter of the zone of inhibition for each antimicrobial agent. The results can be presented in a table and then visualised using a bar chart, with the type of antimicrobial agent on the x-axis and the mean diameter of the zone of inhibition on the y-axis. A larger zone of inhibition indicates a more effective antimicrobial agent against the specific bacterium tested. Evaluate potential sources of error, such as uneven spreading of bacteria or inaccurate disc placement, and discuss their impact on the results.

Principles of Chromatography

Chromatography is a separation technique that relies on the differential distribution of components between a stationary phase and a mobile phase. The stationary phase does not move, while the mobile phase moves through the stationary phase, carrying the components of the mixture with it. In paper chromatography, the stationary phase is the paper itself and the mobile phase is a solvent. Components that are more soluble in the mobile phase and less strongly adsorbed by the stationary phase travel further up the paper. This allows for the separation of mixtures like inks, food colourings, and plant pigments. The resulting separated components on the paper create a chromatogram.

Performing Paper Chromatography Safely

To perform paper chromatography: draw a pencil line (baseline) near the bottom of a chromatography strip, as pencil is insoluble in most solvents. Spot the mixture to be separated onto the pencil line. Place the paper in a beaker with a small amount of solvent (mobile phase), ensuring the solvent level is below the baseline. Cover the beaker to prevent solvent evaporation. Allow the solvent to move up the paper, carrying the sample components. Once the solvent front is near the top, remove the paper and mark the solvent front with a pencil. Allow the paper to dry. Safety points include handling volatile solvents in a fume cupboard or well-ventilated area, wearing eye protection to prevent splashes, and avoiding skin contact with solvents.

Identifying Substances and Rf Values

After drying, the separate components will appear as distinct spots on the chromatogram. Known pure substances can be run alongside the unknown mixture to identify components by comparing the positions of the spots. The retention factor (Rf value) can also be calculated, which is the ratio of the distance travelled by the spot to the distance travelled by the solvent front. Rf = (distance travelled by spot) / (distance travelled by solvent front). Rf values are characteristic for a given substance under specific conditions (same stationary phase, mobile phase, and temperature) and always have a value between 0 and 1. This value can help identify unknown components, especially when compared to reference Rf values.

Planning, safety and evaluation

Practical planning must identify the independent variable that is changed, the dependent variable that is measured and important control variables kept constant. Follow a repeatable method and record units. Complete a risk assessment: identify each hazard, explain the risk and use a suitable safety control. Repeat measurements, calculate a mean, plot an appropriate graph and evaluate anomalies, uncertainty and limitations before suggesting justified improvements.

Practical planning must identify the independent variable that is changed, the dependent variable that is measured and important control variables kept constant. Follow a repeatable method and record units. Complete a risk assessment: identify each hazard, explain the risk and use a suitable safety control. Repeat measurements, calculate a mean, plot an appropriate graph and evaluate anomalies, uncertainty and limitations before suggesting justified improvements.

Practical planning must identify the independent variable that is changed, the dependent variable that is measured and important control variables kept constant. Follow a repeatable method and record units. Complete a risk assessment: identify each hazard, explain the risk and use a suitable safety control. Repeat measurements, calculate a mean, plot an appropriate graph and evaluate anomalies, uncertainty and limitations before suggesting justified improvements.

Practical planning must identify the independent variable that is changed, the dependent variable that is measured and important control variables kept constant. Follow a repeatable method and record units. Complete a risk assessment: identify each hazard, explain the risk and use a suitable safety control. Repeat measurements, calculate a mean, plot an appropriate graph and evaluate anomalies, uncertainty and limitations before suggesting justified improvements.

Practical planning must identify the independent variable that is changed, the dependent variable that is measured and important control variables kept constant. Follow a repeatable method and record units. Complete a risk assessment: identify each hazard, explain the risk and use a suitable safety control. Repeat measurements, calculate a mean, plot an appropriate graph and evaluate anomalies, uncertainty and limitations before suggesting justified improvements.

Practical planning must identify the independent variable that is changed, the dependent variable that is measured and important control variables kept constant. Follow a repeatable method and record units. Complete a risk assessment: identify each hazard, explain the risk and use a suitable safety control. Repeat measurements, calculate a mean, plot an appropriate graph and evaluate anomalies, uncertainty and limitations before suggesting justified improvements.