Required Practicals

• Factors that affect enzyme activity:

  • Enzyme concentration
  • Substrate concentration
  • Temperature
  • pH
• How to set up a control in a practical measuring enzyme activity:

  • Replace the enzyme solution with distilled water or boiled enzyme solution
• How to use the results of the practical measuring enzyme activity to find the initial rate of reaction:

  • Plot results on a graph of ‘rate of reaction’ against ‘time’
  • Draw a tangent at time = 0 to find the initial rate
• Practical procedure to measure the effect of temperature on enzyme activity, using trypsin and milk:

  • Immerse equal volumes of trypsin and milk in different test tubes in a water-bath for 5 minutes
  • Mix together and immediately start timing
  • Record the time taken for the milk to be completely hydrolysed
  • Test at least 5 temperatures, with at least 3 repeats at each temperature
• How to calculate the rate of reaction from time:

  • Rate of reaction = 1/time
• Effect of temperature on enzyme activity:

  • As temperature increases, kinetic energy increases leading to more ES complexes formation
  • Rate of reaction increases up to the optimum temperature
  • Beyond the optimum temperature, bonds in the enzyme tertiary structure break, changing the shape of the active site
  • Substrate and enzyme are no longer complementary, leading to a decrease in the rate of reaction
• Risk associated with handling enzymes:

  • Students may have allergic reactions to enzymes
  • Avoid contact with skin and eyes, wear eye protection
  • Level of risk: Low
• Cells undergoing mitosis in plants can be found in meristem tissue at shoot and root tips
• The mitotic index is the ratio of cells undergoing mitosis to the total number of cells in a sample
• Procedure to prepare a root tip slide:

  • Warm 1M HCl to 60°C in a water bath
  • Cut a root tip using a scalpel and add to the HCl. Leave for 5 minutes
  • Remove from HCl and wash with distilled water
  • Cut the tip of the root tip sample and place on a slide
  • Add a few drops of stain to make chromosomes visible
• Formula for the mitotic index: Mitotic index = Number of cells with visible chromosomes / Number of cells in sample
• Hazards and precautions for reagents used in this procedure:

  • HCl: corrosive, avoid contact with skin, wear eye protection
  • Toluidine Blue O stain: irritant, avoid contact with skin, wear eye protection
  • Scalpel: cut away from fingers
• Purpose of calibration curves:

  • Used to determine the concentration of an unknown sample by comparing it to a set of standard values with known concentrations
• How to find the concentration of plant tissue using a calibration curve:

  • Plot a calibration curve of percentage change in mass against concentration
  • Find the x-intercept where the plant tissue is isotonic to the sucrose solution
• Effects of placing plant tissue in a hypotonic solution:

  • Water moves into the plant tissue by osmosis
  • Plant tissue increases in mass
• Effects of placing plant tissue in a hypertonic solution:

  • Water moves out of the plant tissue by osmosis
  • Plant tissue decreases in mass
• Reason for leaving potato discs in solution for 20 minutes:

  • To allow time for osmosis until the plant tissue reaches equilibrium with its surrounding solution
• Water potential is determined by:

  • The concentration of solutes
  • The higher the solute concentration, the lower the water potential
• Procedure for investigating osmosis using potato tissue:

  1. Make a simple dilution of 1M sucrose to produce 5 concentrations. Add 5 cm3 to 5 different test tubes.

  2. Cut a potato into equal sized chips and weigh.

  3. Place a chip in each test tube and leave for 20 minutes.

  4. Take out, dab the excess water, and weigh them again.

  5. Calculate the percentage change in mass
• Reason for using percentage change rather than the actual change in mass:

  • Potato chips may not all have the same starting mass
  • Percentage change allows comparison
• Significance of the x-intercept of the calibration curve:

  • Indicates the concentration that is isotonic to the solution tested
• Explanation of the change in mass in the potato chips:

  • Chips with a concentration lower than the sucrose solution lose mass due to a net movement of water out of the cells
  • Chips with a concentration higher than the sucrose solution gain mass due to a net movement of water into the cells
• Purpose of dabbing potato chips dry after removing from the sucrose solution:

  • To remove any excess water clinging to its surface
• Controlled variables of this practical:

  • Volume of sucrose solution
  • Size of potato chips
  • Length of time left in solution
  • Dab each potato disc with paper towels
• Factors that affect the permeability of cell membranes:

  • Temperature
  • Concentration of solvents (ethanol)
• Beetroot is used to measure the permeability of cell membranes by observing the amount of red pigment that leaks out into the surrounding solution within a given time. A colorimeter can be used to determine the absorbance and concentration of the pigment
• Procedure to investigate the effect of temperature on permeability of cell membrane:

  1. Cut beetroot into 6 identical cubes with a scalpel

  2. Place each cube in a different test tube with equal volumes of distilled water

  3. Place each test tube into water baths ranging from 30-80°C and leave for 20 minutes

  4. Filter each solution out into a cuvette and measure the absorbance using a colorimeter
• Safety hazards involved in testing the effect of ethanol concentration on membrane permeability:

  • Ethanol is an irritant and flammable, keep away from naked flames and wear eye protection
  • Keep sharp scalpel away from fingers
  • Handle hot liquid with care
• Effect of temperature on membrane permeability:

  • Increasing temperature results in increased membrane permeability
• Effect of ethanol concentration on membrane permeability:

  • Increasing ethanol concentration leads to increased membrane permeability
• Label lines in a diagram should be drawn with a ruler, no arrows, without crossing other label lines, in pencil
• A diagram should be drawn large - at least half the space given

  • No shading
  • Single continuous lines (no sketching)
  • With pencil
• 6 aseptic techniques:

  • Wipe down surfaces with antibacterial cleaner, before and after experiment
  • Use a Bunsen burner in the work space so that convection currents draw microbes away from the culture
  • Flame the wire hoop before using it to transfer bacteria
  • Flame the neck of any bottles before using them to prevent any bacteria entering the vessel
  • Keep all vessels containing bacteria open for the minimum amount of time
  • Close windows and doors to limit air currents
• Bacteria is incubated at 25°C to prevent the growth of pathogens (harmful bacteria), which occurs at higher temperatures
• To compare the effectiveness of different antibiotics applied to the same bacteria, measure the diameter and calculate the area of the zone of inhibition (clear zone) on the agar
• The zone of inhibition indicates the bacteria killed by the antibiotic. The larger the zone, the more effective the antibiotic. If an antibiotic has very little or no zone of inhibition, the bacteria is likely resistant to the antibiotic
• Hazards and precautions of this practical:

  • Naked flame: keep away from flammable materials, tie hair up, wear goggles
  • Bacteria is a biohazard, use disinfectant and wash hands, dispose of bacteria safely
  • Disinfectant is flammable, keep away from naked flame
• The lid should not be completely taped to the petri dish to allow oxygen to enter, preventing the growth of harmful anaerobic bacteria
• A bar chart of zone of inhibition against antibiotic can be plotted from the results of this practical
• The purpose of chromatography is to separate different components in a sample