Practicals

11. Investigate photosynthesis using isolated chloroplasts - Topic 5

Cut 3 leaves of the same species with scissors discarding the stalks and skeleton Place in a cold mortar with 20cm3 of cold isolation medium and grind vigorously Place a layer of muslin around a funnel and pour ground leaf into it and squeeze out the liquid into centrifuge tube (all tubes should have the same volume) Centrifuge tubes at 3500rpm for 3 mins Pour off supernatant, not loosing the pellet. Re-suspend the pellet in 2cm3 of isolation medium and squirt it in and out of pipette to give a uniform suspension Store in an ice water salt bath Tube 1; extract and DCPIP, 2;isolation medium and DCPIP, 3; extract and DCPIP in dark, 4; extract and distilled water, 5; supernatant and DCPIP When DCPIP is added shake tubes, note time and wrap 3 in tin foil. Place all tubes 15cm from bright light, after 3 mins note any changes

5.Prepare and stain a root tip squash to observe the stages of mitosis - Topic 3

Cut 5mm off of garlic root tips (white healthy tips) Soak tips in 2cm of 1M hydrochloric acid for 5 minutes Put tips in 5cm of distilled water for 4-5mins transfer one of the tips to a microscope slide Break up the tip with a mounted needle add a drop of toluidine blue and leave for 2 mins cover with coverslip and blot with several layers of tissue and press gently to spread cells view under microscope fot cell with visible chromosomes Repeat and calculate mitiotic index or the number of cells in a certain phase

14. Use gel electrophoresis to separate DNA fragments of different lengths - Topic 6

DNA placed in a thermocycler with nucleotides, primers and taq polmerase. DNA heated to 94 for 1 min to break H bonds Temp reduced to 54, bonds form Temp increased to 72, bases placed in correct position Mix DNA with resitriction enzyme and loading dye Prepare agar and place into electrophoresis mould Fill electrophoresis tank with buffer solution Use micropipette to put DNA samples into wells, connect to electric supplies and turn on and leave until dye has moved enough Compare the stands to see if any match

1. Investigate the effect of caffeine on heart rate of Daphnia - Topic 1

Daphnia of same species and volume Placed on microscopic tray in pond water in bits of cotton and allowed to acclimatise for 5 minutes Then HR counted using tally marks of a sheet of paper for 15 seconds, x4 to give hr/min repeated with unknown conc. of caffeine (blind) 0.0% and distilled water, 0.1% and 0.5% Repeat with each daphnia and with other daphnia calculate mean

8. Determine the tensile strength of plant fibres - Topic 4

Use celery and flax plants Strip the xylem vessels from the 2 plants measure a standard length (10cm) and cut them all to the same length. Tie each end onto the clamp and move the clamps apart so the xylem is straight but not stretched Add 10g to the middle of the xylem using a hook. Go up in 10's up to 100g then up in 20g's until the fibre snaps Repeat at least x5 for each species.

7. Investigate plant mineral deficiencies - Topic 4

Fill 5 beakers with pearlite (10g) Make a solution of 10cm3 distilled water that contains all nutrients (nitrogen, magnesium, calcium)-positive control Create the solution again but with just distilled water-negative control Make the solution again with one of the 3 minerals missing Pour each solution into one beaker with pearlite Add 5 seeds with the roots pointing down to each beaker and cover with clingfilm leaving small gaps for air Leave for 5 days under red light in a water bath at 20oC Measure shoot growth and observe differences

12. Investigate the effect of temperature on the initial rate of an enzyme-catalysed reaction, to include Q10 - Topic 5

Fill a burette with water and invert in water bath, ensure meniscus is within measurement section Record the starting volume Remove 3 pear chunks of the same volume and s.a. from a boiling tube within water bath Crush using mortar and pestle and transfer to boiling tube, put in water bath allow to reach same temp. Place end of rubber delivery tube in in burette so gas is collected Measure 5cm3 of hydrogen peroxide and place into boiling tube, immediately start timer and place bung firmly on boiling tube Take a reading every 10secs up to 5 mins and mark with a pen Repeat x5 for each temp with at least 5 temps (0,10,20,30,40,50,60)

9. Investigate the antimicrobial properties of plants, including aseptic technique for the safe handling of bacteria - Topic 4

Melt 15cm of sterile agar by placing in a water bath using a septic technique and a blue flame, wave the lid of the agar through the flame and the tube of the bacteria, then using a clean packed pipette squirt 1cm of bacteria into the agar bottle close the lid and mix. Then pour then agar bacteria into a petri dish and leave to harden Crush up plant extracts and mix with methylated spirit, place the liquid of the different plants onto sterile antibiotic disc papers, let them dry for 10 minutes then place the disks onto the agar using sterile forceps. Incubate plates for 24 hours at 25. Observe plates without opening. and calculate zone of inhibition.

4. Investigate the effect of enzyme and substrate conc. on the initial rate of reactions - Topic 2

Pipette 8cm3 trypsin and 2cm3 of distilled water into a boiling tube (0.4%), then 6, 4 (0.3%), then 4, 6 (0.2%) Use a colorimeter and fill with distilled water in a curvette and set to % transmission at 440nm filter Pipette 2cm3 of of milk into beaker, then 2cm3 of 0.5% trypsin into into another beaker Transfer milk into trypsin and immediately transfer to curvette, place in colorimeter and press 'T' record results to 2 dp record % transmission every 10 secs, stop at 3mins Repeat for all conc. 0.5-0.2

16. Investigate rate of respiration - Topic 7

Place 10cm of potassium hydroxide in a boiling tube Place 12 maggots (same mass in total) into wire mesh and place in boiling tube above acid Place both bungs on the U shaped respirometer at the same time ensuring level of red dye does not leave tube. Close the oxygen tap and mark starting position with a marker, then mark where the red dye moves to once every minute for 5 mins Calculate the volume of oxygen moved Repeat and calculate mean rate of oxygen uptake

13. Investigate the effects of temperature on the development of organisms (seedling growth rate) - Topic 5

Set up 7 incubators (0,10,20,30,40,50), Place a lamp above each tray. Set up 7 soil trays filled with pearlite and mineral solution with 25 seeds per tray Leave seeding in sealed incubated container for 2 weeks adding 5cm3 of water each day at the same time Measure the number of seeds that have germinated and the length they have grown (for shrimp hatching, change temp in water bath and use control number of eggs and conc. of water contents)

2. Investigate the vitamin C content of food and drink - Topic 1

Use Titration Pipette 5cm3 of DCPIP into a conical flask and place on a white tile under burette (filled with juice) Read start volume of juice Add juice drop by drop swirling conical flask unitil blue colour of DCPIP turns colourless Subtract start volume of juice from final volume Repeat with same juice then with other juice Using a standard curve calculate conc. of vitamin C

17. Investigate the effects of exercise on tidal volume, breathing rate, respiratory minute ventilation and oxygen consumption using data from spirometer traces - Topic 7

Use a spirometer which is attached to a person, they have a nose clip on and breath into the chamber. Soda lime absorbs CO2 so only oxygen uptake is measured. A person is attached to the machine and does excersize for x amount of time, their breathing rate can be calculated as well as their vital capacity (max. volume of air can be breathed in and out of lungs in one breath), tidal volume (volume of air breathed in and out in one breath at rest), minute ventilation (tidal volume x breathing rate)

15. Investigate the effect of different antibiotics on bacteria - Topic 6

Use aseptic technique Prepare an agar plate with a known bacteria using aseptic technique Flame forceps and use them to pick up a pre-prepared antibiotic disk/mast ring Place firmly in the centre of the agar and tape dish using 2 bits of tape Keep it upside down at 25oC for 2 days Measure zone of inhibition, larger zones = better antibiotic

3. Investigate the membrane structure and the effect of temperature on membrane permeability - Topic 2

Use beetroot of the same species or plant cut cylindrical samples using a size 4 cork bore, then cut into 1cm sections Place in distilled water overnight Labelled boiling tubes filled with 5cm3 of distilled water into water baths (0,10,20,30,40,50,60,70) Place one beetroot section into each tube and leave for 30mins Carefully remove beetroot(no squeezing) and shake water to disperse dye Put 2cm3 distilled water into curvette and put in colorimeter set to %absorption on blue/green filter Adjust to 0 for distilled water Add 2cm3 of of dye, not changing any settings and read % absorbency

18. Investigate habituation to a stimulus - Topic 8

Using a giant African land snail and place it on a clean slightly wet surface, wait until fully emerged. Damp a cotton wool bud and firmly touch the snail between the eyes and immediately start to stopwatch Measure the length of time it takes for the snail to fully extend. Repeat for 10 touches with at least 5 snails

10. Carry put a study into the ecology of a habitat (quadrats and line transacts) to determine distribution and abundance of organisms and to look at abiotic factors - Topic 5

Visited Juniper Halls Used line transacts and quadrats to look at the species present going from a path into a woodland Measured light intensity, soil moisture

6. Identify sclerencyhma fibres, phloem sieve tubes and xylem vessels and their location within stems through a light microscope - Topic 4

cut a stem widthways into a thin strip, place on a microscope slide with a cover slip ontop Place under microscope Complete a high power and low power drawing of the vascular bundle