Module 1

• Types of sampling


  • Random
  • Non-random
  • Stratified
  • Opportunistic
• Random sampling


  Sample sites are selected randomly, e.g. using random numbers as coordinates
• Random sampling


  • Prevents selective sampling, ensuring the data is not biased
  • Not all areas/types of habitat may be sampled equally, leading to inaccurate data. Also, species with a small population may be missed, leading to an underestimate of biodiversity
• Stratified sampling


  The habitat is divided into groups that appear different, and each area is sampled separately. Each group should be mutually exclusive and collectively exhaustive.
• Stratified sampling


  • Ensures that all areas are sampled and prevents small populations from being missed out
  • May lead to some over-representation of some areas in the study, for example small areas may be sampled more than necessary because they look different from other areas
• Opportunistic sampling


  Areas are chosen to sample by the researcher either using their prior knowledge, or choosing areas that interest them as they are collecting data
• An example of stratified sampling is if gorse patches are sampled separately from patches of bracken
• Stratified sampling allows areas of different population levels in the same habitat to be sampled equally (e.g. an area with a higher population could be sampled with more transects in the same area)
• Habitat is divided into groups that appear different, and each area is sampled separately
• Mutually exclusive groups


  • Each population can only be in one group
• Collectively exhaustive groups


  • No population must be left out
• Ensures that all areas are sampled and prevents small populations from being missed out (e.g. in random sampling)
• Allows areas of different population levels in the same habitat to be sampled equally (e.g. an area with a higher population could be sampled with more transects in the same area)
• May lead to some over-representation of some areas in the study, for example small areas may be sampled more than necessary because they look different from other areas
• Opportunistic sampling


  • Easier and quicker than random sampling, and produces more data
  • Potential for bias, as species which are more noticeable may be included more often than less noticeable species, leading to an overestimation of their importance, and an inaccurate estimate of biodiversity
• Systematic sampling


  Samples are taken at set distances (e.g. every 5 metres), often using a transect
• Systematic sampling


  • Good to view a change in biodiversity or populations along a line
  • Risk of missing species as only a small area is sampled, which could cause biodiversity to be underestimated
• Calculation of species diversity

  1. Select a suitable area for sampling

  2. Place two long tape-measures at right angles to each other along the border of the region you are going to sample

  3. Randomly generate numbers to decide where to sample

  4. Place the left-hand corner of the quadrat at the position where the two coordinates meet/intersect

  5. Identify the different species in the quadrat using a key and count the number of each present. Calculate the percentage cover

  6. Generate new coordinates and repeat the sampling process at least 9 times more

  7. Use Simpsons index of diversity to calculate the species diversity (D)
• Calculation of species abundance

  1. Perform steps 2-6 of the species diversity method

  2. The population size of each species can be estimated by multiplying up the sample size

  3. Produce a graph of species abundance in different areas
• Calculation of species abundance and distribution

  1. Use a measuring tape to make a transect over the area you wish to sample

  2. Place quadrats at given intervals along the tape measure

  3. Identify the different species in each quadrat using a key and count the number of each present. Calculate the percentage cover

  4. Produce a graph of species distribution against distance along transect (e.g. a kite diagram)
• Investigating the effect of cutting off roots on a plant

  1. Cut a shoot underwater to prevent air from entering the xylem

  2. Cut the shoot at a slant to increase the surface area available for water uptake

  3. Assemble the potometer in water and insert the shoot under water, again to prevent air from entering

  4. Remove the apparatus from the water but keep the end of the capillary tube submerged in a beaker of water

  5. Check the apparatus is watertight and airtight, using screws or petroleum jelly

  6. Dry the leaves

  7. Allow time for the plant to acclimatise and then shut the tap

  8. Remove the end of the capillary tube from the beaker of water until one air bubble has formed, then put the end of the tube back into water

  9. Record the starting position of the air bubble

  10. Start a stopwatch and record the distance moved by the bubble at regular time intervals

  11. Calculate the rate of air bubble movement by dividing the distance travelled by time
• This is an estimate of the transpiration rate
• OCR (A) Biology A-level Module 1: Development of practical skills in Biology
• PAG 6: Chromatography OR Electrophoresis
• Chromatography


  An analytical method used to separate a mixture into the different biological molecules
• Stationary phase


  TLC plate or chromatography paper
• Mobile phase


  The solvent for the biological molecules
• Adsorption


  Where molecules bond to the surface of a substance
• Chromatography - chlorophyll pigment method

  1. Grind up the leaves using a pestle and mortar, with some anhydrous sodium sulphate and then add some propanone as a solvent

  2. Transfer the liquid to a test tube and add some petroleum ether and shake

  3. Transfer some liquid from the top layer with some anhydrous sodium sulphate

  4. Draw a line on the TLC plate with a pencil about 2cm from the bottom

  5. Use a capillary tube to take up some of the pigment solution, and then use this to place a dot of pigment in the middle of the pencil line

  6. Allow the dot to dry, then add another spot over the first dot. Repeat this until you have a concentrated spot of pigment

  7. Add a small amount of solvent in a beaker (less than 2cm depth – the pencil line should be above the solvent line)

  8. Place the TLC plate in the beaker with the pencil line towards the bottom

  9. Place a watch glass over the beaker to stop the solvent evaporating

  10. When the solvent almost reaches the top, take the paper out and mark the solvent front with a pencil. Also mark at the side of the plate the locations of the pigments

  11. Leave the paper to dry

  12. Measure with a ruler the distance travelled by each pigment up the plate, as well as the distance the solvent moved

  13. Calculate the Rf value for each spot by dividing the distance travelled by the pigment by the distance travelled by the solvent
• Chromatography - amino acid method

  1. Draw a pencil line near the bottom of a piece of chromatography paper

  2. Put a concentrated spot of the mixture of amino acids on the paper

  3. Add a small amount of solvent (butan-1-ol, glacial ethanoic acid and water) to the beaker. Put the chromatography paper in and ensure the pencil line is above the solvent line

  4. Place a watch glass over the beaker to stop the solvent evaporating

  5. When the solvent almost reaches the top, take the paper out and mark the solvent front with a pencil

  6. Leave the paper to dry

  7. Spray the amino acids with ninhydrin solution, which makes them go purple

  8. Measure the distance the solvent moved and the distance each spot moved up the plate

  9. Calculate the Rf value for each spot by dividing the distance travelled by the spot by the distance travelled by the solvent

  10. Compare experimental values to known values to identify the unknown amino acids
• Electrophoresis


  1. The DNA first has to be cut into fragments by restriction enzymes

  2. Pour agarose gel into a gel tray a leave it to solidify

  3. A row of wells is created at one end of the tray

  4. Put the gel tray into a gel box/tank

  5. Make sure the end of the gel tray with the wells is closest to the negative electrode on the gel box

  6. Add buffer solution to the reservoirs at the sides of the gel box so that the surface of the gel becomes covered in the buffer solution

  7. Using a micropipette, add the same volume of loading dye (10 ul) and DNA sample and put in the bottom of a well

  8. Repeat this process for each DNA sample, using a clean micropipette for each different DNA fragment

  9. Record which DNA sample goes into which well

  10. Put the lid on the gel box and connect the leads from the gel box to the power source

  11. Turn on the power source and set it to 100V

  12. Let the gel run for 30 minutes or until the dye is about 2 cm from the end of the gel

  13. Remove the gel box and tip off any excess buffer solution

  14. Wearing gloves stain the DNA fragments by covering the surface of the gel with a staining solution then rinsing the gel with water
• This method can be used with RNA fragments and proteins too
• The bands of DNA in each sample can be compared for similarities and differences
• PAG 7: Microbial Techniques
• Effect of antibiotics on bacterial growth - method

  1. Wash your hands and disinfect your work area

  2. Have Bunsen Burner on nearby to sterilise the air and prevent air-borne microorganisms settling

  3. When opening the bottle of broth, pass the neck over the Bunsen Burner flame to prevent the microorganisms in the air entering the bottle

  4. Only open a petri dish enough to allow you to introduce your desired organisms

  5. All equipment should be sterilised by passing it over the flame before and after use

  6. Using a sterile pipette, add the same volume (0.1cm3) of each antibiotic to a different Petri dish

  7. Dip an inoculating loop in the broth (the broth of your desired bacteria)

  8. Spread a streak of broth over the agar surface, then close the Petri dish, and tape it shut. Repeat for the remaining Petri dishes

  9. Label each petri dish

  10. Place the petri dishes in a warm incubator

  11. Leave all the plates for the same amount of time (e.g. a day) then observe the results

  12. Count the number of colonies that have formed on each plate and record results in a table

  13. Work out the mean number of colonies formed for each antibiotic
• If colonies overlap, make serial dilutions with the bacteria broth so there is less bacteria initially so less colonies are present and it is more manageable
• PAG 8: Transport In and Out of Cells
• Investigating cell membrane permeability - the effect of temperature on beetroot cell membranes

  1. Use a scalpel to cut five equal sizes of beetroot. Rinse the pieces to remove any pigment released during cutting

  2. Add the 5 pieces to 5 different test tubes, each containing 5 cm3 of water

  3. Place each test tube of in a water bath at different temperatures for the same length of time using a stopwatch

  4. Remove the pieces of beetroot, leaving just the liquid

  5. Carry out colorimetry (use the blue filter)
• Permeability


  The higher the permeability, the more pigment released, the higher the absorbance reading
• Investigating diffusion - Concentration

  1. Make some agar jelly with phenolphthalein and dilute sodium hydroxide

  2. Prepare 5 test tubes containing HCl in increasing concentrations e.g. 0.2M, 0.4M, 0.6M, 0.8M and 1.0M

  3. Using a scalpel, cut out 5 equal size cubes from the agar jelly

  4. Put one of the cubes into the first test tube and use a stopwatch to time how long it takes for the cube to turn colourless

  5. Repeat for the other concentrations, using a new cube each time