Core Practical Experiments

Unit 2

Root Tip Squash

Procedure:
1. Carefully cut the apical 5mm from the tip of a growing lateral root
for example an onion.
2. Place the root tip in a watch glass containing acetic orcein and 1.0
mol dm of hydrochloric acid in the approximate proportions of 10
parts stain to 1 part acid.
3. Warm, but do not boil for 5 minutes on a hotplate or by passing it
repeatedly through a low Bunsen flame.
4. Place the fixed root tip on a microscope slide and add 2 drops of
acetic orcein.
5. Without interfering too much with the arrangement of the cells,
break the root tip with a mounted needle so as to spread the tissue
out as thinly as possible.
6. Put a cover slip over the tissue, cover it with a filter paper to absorb
the excess stain and squash it gently by pressing down on the cover
slip using your thumb. Take care to avoid lateral movement and
don’t break the cover slip.
7. Warm the slide on a hotplate for 10 seconds.
8. The slide should be hot but not too hot to touch.
9. Carefully observe the slide using a light microscope and identify the
stages of mitosis.

Why is the root tip used?

- This is the region of active cell division by mitosis

Why is hydrochloric acid used?

- breakdown the pectins in the middle lamella that hold the
cells together. Helps produce a preparation that is one cell
thick.

Why is the stain used?

- makes chromosomes more visible

Why do we press down on cover slip?

- forms a thin layer of cells with no overlapping cells present.

Why do we heat?
- Helps intensify the stain.

Totipotency and Plant Tissue Culture

Procedure:
 1. Sprinkle some seeds of white mustard onto damp cotton placed on
a plastic tray.
2. Cover with transparent cling film and place in a warm, bright place
to germinate. When the seedlings have just started to unfold their
cotyledons, they are ready to be cultured.
3. Measure out 2.5g of agar powder and add to 250cm of distilled
water. Boil and stir gently with a glass rod until the agar dissolves.
4. Whilst the agar is still molten, pour about 2cm depth into several
short-necked test tubes. Allow cooling and solidifying.
5. With a sharp pair of scissors, cut the tops of the seedlings just
below the shoot apex.
6. These are the explants. Leave the hypocotyls (the early stem) and
roots behind on the sponge.
7. Carefully push the cut end of the explant into the agar. Put one
explants into each specimen tube. Make sure the cotyledons do not
touch the agar.
8. Cover the tubes with cling film. Place tubes on sunny windowsill.
9. Observe the progress of your explants daily and record when
anything of note develops. Observe over a period of 10 days.

Systematic errors
- errors in calibration of measuring instrument

Random errors
- possible contamination of plant by microorganisms
- temperature fluctuations
- inconsistent light intensity

Increasing Reliability of Experiment

- repeat at least 3 times
- repeat/ignore anomalies

Use of agar
- water source and support

Why short-necked test tubes?

- easier to insert explant

Cover with cling film?

- prevent contamination / loss of moisture

Why not open tubes once they have been set up?
- prevent contamination by pathogens
-

Tensile Strength of Plant Fibres

Procedure:
1. Measure the diameter of the plant fiber using a vernier caliper and
calculate the cross-sectional area of this plant fiber.
 2. Measure the length of the plant fiber
3. Clamp the fiber at each end securely
4. Add on slotting weights of increasing mass to this fiber until it
breaks.
5. Take note of the total mass that causes the fiber to break and
convert mass into force by multiplying the mass in kg with 10.
6. Tensile strength is calculated by dividing force (N) by the cross-
sectional area of the fibre (m )

Tensile strength = Force / Cross sectional area of fiber

7. Repeat this procedure using fibers from different plants and

compare their tensile strength.
8. Note : Plant fibers are obtained via retting whereby stems are
immersed in water. During soaking, bacteria and fungi break down
the soft tissues of the stems leaving the cellulose fibers intact.

Systematic errors – errors in calibration of measuring instrument

Random errors
- water content of fibres
- temperature fluctuation of environment
- humidity level of environment

To improve reliability
- repeat experiment 3 times
- control the variables

Risks
- when plant fibre snaps, masses can drop fast. Place mat on
table so masses drop on mat.

Plant Mineral Deficiencies

Procedure:
1. Using a measuring cylinder, half fill a tube with the “all nutrient
present” solution.
 2. Cover the top of the rube with foil and push down on the covering
so there is a ‘well’ in the centre. Make a small hole in the foil.
3. Gently push the roots of a radish/barley/maize plantlet through the
hole so it is in the solution below.
4. Repeat these steps using the other solutions:-
a. Solution lacking nitrogen
b. Solution lacking phosphate
c. Solution lacking magnesium
d. Solution lacking calcium
e. Solution lacking all nutrients

5. Ensure that the measuring cylinder is rinsed out between solutions.

6. Wrap the tubes in black paper to exclude light from the solutions
and to prevent the growth of algae.
7. Place the tubes on a sunny windowsill.
8. Observe regularly, top up the solutions as needed and note any
changes.
9. Make any appropriate measurements to allow comparisons of the
treatments.

Systematic Errors
- calibration errors in measuring instrument

Random Errors
- temperature fluctuations
- humidity levels
- light intensity
- [Oxygen] and [carbon dioxide] in environment

Increasing reliability
- repeat at least 3 times
- control variables

Anti-Microbial Properties of Plants

Procedure:

Part One
 1. Liquefy the nutrient agar by placing the bottle in a water bath at
100 .
2. Once the agar has liquefied, remove the bottle and loosen the cap
to allow air to escape.
3. Allow the agar to cool to about 45 .
4. Using a syringe, withdraw 1cm of bacterial broth and place it into a
sterile Petri dish using aseptic techniques.
Aseptic Techniques
 Wipe the workbench with a disinfectant
 The lid of the dish is only lifted at a 45 angle to only allow
entry of the syringe
 Procedure is carried out near a Bunsen flame

5. Pour the molten agar into the Petri dish so that it is half-filled.
6. Very gently push the Petri dish back and forth to uniformly mix the
bacteria and the agar.
7. Allow the agar to set by leaving it aside for about 20 minutes.

Part Two
1. Obtain a plant extract by crushing 3g of plant material with 10cm of
industrial denatured alcohol and shake it from time to time for 10
minutes.
2. Use a syringe and withdraw 0.1cm of the extract onto a sterile disc
cut out from a filter paper.
3. Let the paper discs dry for 10 minutes on an open sterile Petri dish.
4. Repeat steps 1-3 for other plants, making separate test discs for
each extract.
5. Prepare one disc to act as a control.
6. Using sterile forceps, place the test discs together with the control
disc onto the medium in the Petri dish.
7. Ensure that you can distinguish between the different discs by
making the underside of the Petri dish.
8. Close the Petri dish and tape it to secure the lid with adhesive tape
at 4 corners.
9. Incubate the dish for 24 hours at 30 .

Note : Make sure to wash your hands.

Systematic error
- error in calibration of syringe used

Random error
- possible contamination of microorganism
- nutrient level in nutrient agar could have been diminished

UNIT 1
Effect of Caffeine on Heart Rate
Procedure:
1. Place a few strands of cotton wool on a cavity slide to restrict the
movement of the water flea.
2. Using a pipette, transfer one large water flea to a cavity slide.
3. Remove the water from around the water flea using filter paper,
then add one or two drops of distilled water or pond water.
4. View the water flea under the low power and focus on its heart
which can be seen through its translucent body.
5. Use a stopwatch to record the number heartbeats per minute. In a
pair, one person counts the beats while the other person observes
the time period.
6. Tap a pencil on a piece of paper and count up the pencil marks at
the end of the period.
7. Do a ‘blind’ study so the person counting the number of heartbeats
is not biased.
8. Repeat the procedure using other water fleas from the culture
solution on fresh, clean slides. Replace the water with caffeine
solution.

Constant : temperature & size of Daphnia

Reliability – repeats

Validity – same Daphnia used

Explanation :
- caffeine stimulates sympathetic nerves to transmit impulses
at a high frequency to the heart which causes an increase in
heart rate

Why is Daphnia used?

- Not an endangered species
- Bred for fish food and so will die either way
Vitamin C content of Fruit Juice
Procedure:
1. Prepare standard solutions containing respectively 0.5, 1.0, 2.0 and
4.0 mg cm of Vitamin C (ascorbic acid)
2. Using a syringe, place 2 cm of DCPIP solution in a test tube.
3. Fill a 1 cm syringe with 4.0 mg cm Vitamin C solution. Add this
solution drop by drop to the DCPIP solution, stirring gently with a
glass rod after each drop.
4. Determine the number of drops needed to decolorize the DCPIP
solution.
5. Repeat this procedure using fresh samples of DCPIP each time, with
the other 3 solutions of Vitamin C and finally with the orange juice
and pineapple juice.

How to increase accuracy?

- colorimeter to determine end point
- use burette to drop fruit juice and DCPIP

Effect of Temperature on membrane structure of beetroot

Procedure:
1. Using a sharp scalpel cut twenty-five discs from a beetroot rod,
each approximately 2mm thick.
2. Prepare 5 water baths. Bring the temperature of the water to 10 ,
40 , 50 , 60 , and 70 .
3. Place 5 boiling tubes each containing 5 cm distilled water into the
water baths of the specific temperatures.
4. Leave for 5 minutes until the water reaches the required
temperature.
5. Place 5 discs into each of the boiling tubes. Leave for 30 minutes in
the water baths.
6. Remove beetroot sections; shake the water/solution to disperse the
dye.
7. Switch on the colorimeter and set it to read % absorbance.
8. Set the filter dial to blue/green filter.
9. Using a pipette, measure 2 cm distilled water into a cuvette. Place
the cuvette into the colorimeter; making sure that the light is
shining through the smooth sides.
10.Adjust the colorimeter to read 0 absorbance for clear water. Do not
alter
the setting again during the experiment.
11.Place 2 cm of the dye solution into a colorimeter cuvette and take
a reading
for absorbency.
12.Repeat the readings for all the temperatures.
Effect of enzyme concentration on the activity of amylase
Procedure:
1. Prepare 5 different concentrations of the enzyme solution
(undiluted, diluted to half, diluted to quarter, diluted to one-tenth of
the original concentrations).
2. Set up a water bath at 35 .
3. Pipette 5cm of undiluted enzyme solution into one test tube and 5
cm of starch solution into another test tube.
4. Stand both test tubes in the water bath and leave for 10 minutes so
that both the solutions reach the same temperature before mixing.
5. Mix the enzyme and starch solutions together, replace the mixture
in the water bath and immediately start a stopwatch.
6. At intervals of 1 minute, remove a drop of the mixture and test it
with iodine solution on a white tile.
7. Continue the experiment until the mixture fails to give a blue-black
colour with iodine solution. Record this as the achromatic point.
8. Repeat this procedure with the other concentrations of amylase.
Use exactly 5 cm of enzyme solution and 5 cm of starch solution.
Effect of substrate concentration on the activity of catalase
Procedure:
1. Add sufficient of the 1.5M hydrogen peroxide solution into a small
beaker to a depth of 4 cm.
2. Using a pair of forceps, dip one of the squares of filter paper into the
potato extract. Shake once to remove the excess fluid.
3. Hold the filter paper 1 cm above the surface of hydrogen peroxide
solution and drop it into the solution.
4. Start a stopwatch as soon as the filter paper touches the bottom of
the container.
5. Stop the timing as soon as the square rises from the bottom of the
container.
6. Remove the filter paper.
7. Repeat this procedure twice using 1.5M solution with a fresh square
of filter paper each time.
8. Repeat steps 1-7 using the other 3 different concentrations of
hydrogen peroxide provided , using a fresh square of filter paper
each time.

Explanation :
- more free substrate molecules, more collisions, more ESC
formed

Random error :
- difficult to determine time when filter paper reaches the top
of the surface
- concentration of potato not standardized

Reliability
- repeat the experiment
- standardize temp, pH
- change hydrogen peroxide solution after every run