As Biology Core Practical Experiments

AS BIOLOGY CORE PRACTICAL EXPERIMENTS

UNIT 1

1. ​The Effect of Caffeine on Heart Rate

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Why Daphnia?

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Daphnia are small and show the results of the experiment quickly

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They have simple nervous systems so are less likely to feel pain

They are abundant so easy to get hold of / No damage to environment when a few are removed from it -

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Transparent so easy to measure heartbeat

Variables to be controlled are:

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Temperature

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Volume of solution

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Time of acclimatisation

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Daphnia must be the same age / size

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Daphnia must have the same health

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Counting time

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Method:

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​Independent variable:​ Caffeine concentration

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​Dependent variable:​ Heart rate of Daphnia

Place a Daphnia in each of 5 different solutions (4 different concentrations of caffeine and one with distilled water to act as a control) -

Leave Daphnia for 5 minutes to acclimatise

Immobilize the Daphnia using a little cotton wool in a cavity slide and observe under microscope -

Count and record the no. of heartbeats in one minute

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Repeat 5 times at each concentration and allow means to be calculated

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Outcome: As caffeine concentration increases, heart rate also increases

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How does caffeine increase heart rate?

Increasing caffeine concentration causes the electrical activity of the sinoatrial node to increase, making it depolarize. As it depolarizes, the right and left atria contract and the impulse travels to the atrioventricular node where, after a delay of about 0.13 seconds, the impulse continues to travel towards the ventricles. This delay ensures that the atria have finished contracting and ventricles are full. The signal then reaches the Purkyne fibres that conduct the impulses to the apex of the ventricles where contraction begins and travels upwards towards the atria.

Caffeine also affects the ventricles, leading to an increase in the rate of contraction and relaxation of each heart beat. This means that, as well as beating faster, the heart's individual beats are associated with an increased cardiac output.

2. ​The vitamin C Content of Fruit Juice

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Variables to be controlled:

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Mass of fruit / age / source / …

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Time for storage

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Method of juice extraction

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Volume / concentration of juice / DCPIP

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Temperature

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Same end point colour

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Method:

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​Independent variable:​ Fruit juice

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​Dependent variable:​ Volume of juice required to decolourise 1cm3 of DCPIP

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Put 1cm3 of DCPIP solution into a test tube

Fill a plastic syringe with juice and add drops to the DCPIP until the blue of the DCPIP is lost. Record the volume of juice added -

Repeat 5 times for each juice to calculate means

To calculate the actual Vitamin C concentration, the DCPIP solution must be calibrated. A solution of known Vitamin C concentration is added to 1cm3 of DCPIP until it is decolourised and the volume recorded.

Conc. of Vitamin C in juice = (Con. of Vitamin C solution x Volume of Vitamin C solution needed to decolourise 1cm3 DCPIP) ÷ Volume of fruit juice needed to decolourise 1cm3 DCPIP

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Limitations: Difficulty in controlling temperature

End point difficult to judge as needs to be just when blue colour disappears especially in highly coloured juices -

Some loss of solution when transferring from one beaker to another

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Accuracy of measuring equipment

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Storing fruit at low temperatures slows down decay because:

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Low temperature reduces / prevents growth of microorganisms

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Low temperature reduces activity of enzymes

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Less kinetic energy means fewer collisions / fewer cell divisions 3. ​The effect of Temperature on Membranes

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Factors that affect the permeability of the beetroot cell membrane are:

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Temperature

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Age

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Storage

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Duration

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Prior treatment with solvent

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pH

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Bile salts

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Variables to be controlled are:

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Source / Species of beetroot

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Age / Size of beetroot

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Volume of water / solution used

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Time left in water or solution

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pH

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Method:

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​Independent variable:​ Temperature of water

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​Dependent variable:​ % transmission of light through resulting solution

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Using a cork borer and knife, cut 5 pieces of beetroot equal in mass and size

Rinse the beetroot pieces with water and gently pat them dry with tissue before using them as, when cutting, pigment is released from broken cells and must be removed before starting or solutions will be darker than they should -

Place one piece into each of 5 tubes and add 5 1cm3 water to each one

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Place each tube into a water bath of different temperature (e.g. 15, 20, 25, 30, 35)

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Leave for 15 minutes

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Remove beetroot and shake tubes to disperse dye.

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Calibrate the colorimeter using distilled water in a cuvette as a reference / control.

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Take readings of absorbance of the water in the tubes

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Take 5 repeats at each temperature and calculate means

· Results and reasons: As temperature increases, % transmission slightly increases. This is due to membrane molecules gaining more heat energy and vibrating more, creating large gaps in the membrane that enable dye to be released. Proteins in membrane may become denatured, leaving large pores through which the dye leaks.

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Limitations:

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Pigment is not equally distributed throughout the beetroot

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Some beetroot may have skin on affecting surface area

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Size of beetroot is difficult to control 4. ​The Effect of Enzyme Concentration on the Rate of Reaction

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Variables to be controlled are:

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Temperature à use a water bath

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Volume of enzyme

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Volume of substrate

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Concentration of substrate

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pH

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Method:

Take 5 test tubes. In 4, place increasing volumes of trypsin solution e.g. (1, 2, 3, 4 cm3 ) and make up the volume to 4 cm3 using distilled water. The other test tube should be filled with 4 cm3 of water to act as a control. -

Add 5 of milk powder (casein solution) as substrate and start the stopwatch

Measure the cloudiness of the solution over time using a colorimeter (every 30 secs for 10 minutes) against water as a reference / control -

Repeat at least 5 times at each concentration and calculate means

· As concentration of enzyme increases, rate of reaction increases up to a point, where all enzyme has metabolised all substrate immediately

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Why do enzymes work better at higher temperatures?

At low temperatures the reaction is slow because the enzyme and substrate molecules don’t collide very often and move slowly. Increasing temperature increases kinetic energy and so frequency of collisions. The substrate binds to the enzyme’s active site more often thus increasing the rate of reaction. After the optimum temperature bonds holding the 3D – shape of the enzyme together start breaking so it loses its shape and the enzyme-substrate complex can no longer form. The enzyme is denatured. UNIT 2

5. ​Observing Mitosis

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Safety:

Risk of injury to hands by sharp knife or mounted needle so wear thick gloves or cut away from body Acid is corrosive so wear gloves to reduce risk of injury and safety glasses to reduce risk of injury to eyes -

Stain may stain clothes and skin so wear gloves and lab coat

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Glass coverslip may break and cut your fingers so wear gloves to protect hands

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Method: Cut the last 0.5 cm off the end of actively growing garlic or onion root tips

Treat with acid to soften tissue by breaking down the middle lamella so that the cells will separate easily when squashed -

Break up the tips gently using a mounted needle on a microscope slide

Add toluidine blue to stain the chromosomes, warming if needed to intensify the stain -

Place a glass coverslip on top and squash gently

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Observe under the microscope

6. ​Totipotency and Plant Tissue Culture

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Safety: Bacteria could grown in agar

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Variables to be controlled:

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Same age / size seeds

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All seeds should come from the same plant

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Temperature

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Light Intensity

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Time allowed to grow

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Atmospheric carbon dioxide concentration

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Humidity

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Method:

Use week-old mustard seedlings. Cut off the top 2cm (stem and leaves) and suspend in agar in a test tube -

Leave for a week and look for new roots / leaves forming

Cells at the bottom of the stem differentiate to become new roots which demonstrates pluripotency

7. ​The Tensile Strength of Plant Fibres

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Safety:

The fibre could enter and injure the eye when it snaps so wear safety glasses to protect eyes Place layers of cloth beneath the mass hanger to stop masses from falling onto the foot and injuring it

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Variables to be controlled:

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Length of fibre

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Size of each individual mass

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Width / diameter / cross-sectional area of fibre

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Fibres must come from the same plant species

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Same age

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Same parent plant to reduce genetic variation

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Same prior treatment

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Temperature

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Humidity

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Method:

Soak nettle plant stems in water for a week to soften the tissues and allow the fibres to be easily extracted Select adequate fibre (taking into account all variables) and attach one end to a clamp and stand then progressively hang masses on the other end -

Record the mass at which the fibre breaks

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Repeat 5 times at each thickness to calculate a mean

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Outcome: Wider fibres are stronger

8. ​Plant​ ​Mineral Deficiencies (Optimum concentration of substances ·

Safety:

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There are no significant safety issues

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Mineral / Plant / Enzyme allergies or irritants

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Hypodronics may provide good growing conditions for bacteria/fungi

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Preliminary Work:

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See if proposed method will work

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See if the plant chosen will grow in hydroponic unit

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Select a range of concentrations

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Check for suitable conditions for digestion e.g. temperature, pH

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Find a suitable method of measuring growth

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Check for most suitable conditions for growth of plants

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Select suitable time scale for measuring growth / stain digestion

· Things affecting enzyme action are: protein type, volume of solution, stirring, pH, temperature, surface area, protein concentration…

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Variables to be controlled:

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Volume of mineral solution

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Concentration of solution

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Species of plant

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Light intensity

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Age / size of plant / seedling

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Genetically similar à Same parent plant

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Temperature

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Method:

​Dependent variable:​ E.g. mass of plant tissue, mass of fruit, length of shoot, number / colour of leaves. Description of method of measuring change in dependent variable ​Independent variable: ​Concentration of magnesium. Range of suitable concentrations suggested (at least 5) (0 (control), 10%, 30%, 50%, 70%, 90%, 100%) Take six plants / seedlings and place each of them into a test tube with a different concentration of solution (one with distilled water to act as a control) Cover each tube with foil to exclude light and prevent algae growth that could affect concentration of mineral ions -

Leave the tubes for a week

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Record changes in mass / height / root length

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Repeat at each concentration / for each mineral ion 5 times and calculate mean

Use of graph to identify other values of concentration to test to identify optimum concentration

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Recordings, Representation and Analysis:

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Table matching method with headings and units.

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Change in growth calculated e.g. by measuring change in length

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Means calculated from repeat data

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Scatter / line graph format with correctly labelled axes

Use of graph to estimate range for optimum / to identify other values of concentration to test to identify optimum concentration Concentrations above those first reaching maximum rate of digestion would be wasteful

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Limitations:

Difficult to control all variables affecting plant growth / protein digestion e.g. seeds do not germinate at the same time, genetic differences between the plants… / surface area of stain, protein concentration -

Limiting factor(s)

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Experimental conditions may not match those normally used

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More than one type of mineral for effective growth of plants

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Difficult to measure the dependent variable 9. ​The Antimicrobial Properties of Plants/Secretions

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Ethical Issues:

Welfare of frogs e.g. frogs should be kept in suitable conditions / not be harmed when collecting secretions

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Return the frogs to their habitat after using them

Avoid skin contact with frogs e.g. wear gloves when handling them / wash hands after handling them / wear eye protection -

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Prevent the growth of harmful bacteria / exposure to harmful bacteria

Safety:

Wipe working area with antiseptic solution / work close to a Bunsen Burner which sets up convection currents of sterile air to prevent growth of unwanted harmful bacteria / contamination Secure lids with cellotape but don’t seal completely in order to avoid pathogenic anaerobic bacteria to grow Don’t use 37 ºC as this is human body temperature and could encourage pathogenic bacteria to grow

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Preliminary Work:

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Practise proposed method / see if proposed method will work

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Allows selection of appropriate species of frog to be worked out

Carry out experiments to determine a suitable method for collecting secretions from frog Carry out experiments to determine appropriate concentration / volume of frog secretion Carry out experiments to determine the most appropriate method of applying the secretions to the plates

Carry out experiments to determine the best parameters for another named variable e.g. suitable timescale for measuring the inhibition of bacterial growth / conditions for growth of the bacteria / type of bacteria / … -

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Determine best method of measuring dependent variable

Variables to be controlled:

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Concentration of plant material / antibiotic

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Lawn of bacteria on petri dish

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Same volume of plant material / antibiotic on each disc

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Disc size

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Bacterial species (E. Coli)

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Temperature

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Method:

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​Dependent variable:​ Zone of inhibition / absorbance of culture

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​Independent variable:​ secretions from different frogs / antimicrobial solution

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Prepare, under sterile conditions, petri dishes with a thin layer of agar in them

Once the agar is set, spread a drop of bacterial culture (E. Coli) over the surface using a sterile glass spreader to form a lawn Prepare the extract by crushing material using a pestle and mortar with alcohol if necessary -

Dip small disk of blotting paper and allow to dry

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Minimally lifting the lid, place on the centre of the agar and press lightly

Secure lids with 2 pieces of cellotape but don’t seal completely in order to avoid pathogenic anaerobic bacteria to grow -

Incubate at 25 / 30 ºC for a week

Observe the plates and the zone of inhibition will be clear. Measure its diameter to give an idea of relative antimicrobial strength / effectiveness against microbes -

Repeats taken and means calculated

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Recordings, presentation and analysis:

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Table which matches method described with headings and units

Change in bacterial growth calculated e.g. by measuring area of zone of inhibition / absorbance of culture -

Means calculated from repeat data

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Graph type selected that matches the data to be collected

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Axes needs to be included

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Appropriate statistical test à t-test / Spearman’s Rank

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Anomalous data ignored.

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Limitations: Difficult to control all variables (affecting bacterial growth)

Other components of secretions may affect bacterial growth masking the effect of the antibiotics

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Difficult to standardise extraction of secretion

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Other variables related to frog e.g. age, size, gender

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Uneven spread of bacteria

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A variable may be acting as a limiting factor for bacterial growth (give example)

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Need to test effect on more than one type of bacteria

A2 BIOLOGY CORE PRACTICAL EXPERIMENTS

UNIT 4

1. ​Study of the Ecology of a Habitat ·

Safety:

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Possible risk of coconuts falling so collect coconuts beforehand.

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Possible risk from indigenous animals unidentified plants, insect bites…

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Possible irritant / allergenic material so wear gloves.

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Working under sun so can be burnt. Wear protection against sunburn.

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Ethical:

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Minimise disturbance to the habitat

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Change in food chain/food web

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Preliminary Work:

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Practice proposed method to see if it will work

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Check for most suitable site/ time of the year

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Check for the most suitable method of measuring yield

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Select suitable intervals of sampling to give sufficient data for analysis

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Consider any other variables that need to be taken into account

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Consider impact of farming practices used like pesticides

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Sampling Methods:

​Random Sampling:​ Used when measuring density of a plant species or slow moving animals. 1. Set up grid using tape measure and use random numbers to generate points to place at least 10 quadrats 2.

Count number of chosen species in each quadrat or estimate % abundance

3. Density = Total no. of plants counted / (Area of one quadrat x Total no. of quadrats taken) ​Systematic Sampling:​ A line transect is used to study changes in plant species across an area. 1.

A tape measure is laid along several zones to be looked

2.

At least 10 quadrats are then placed at regular intervals

3.

Record data

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Variables to be controlled: Abiotic Factors

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Light intensity

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Surrounding vegetation

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Slope

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Temperature

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Soil water

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Humidity

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O2 concentration

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pH

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Record, present and analyse:

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Clear table which matches method description with headings and units

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Means calculated from repeat data

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Scatter/line/bar graph format with correctly labelled axes

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Use of correlation test (Spearman’s Rank, T test)

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Limitations:

Difficult to control all variables (abiotic factors affecting the variables being investigated) -

Difficult to standardise measurement of yield

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Difficult to harvest coconuts high on palm

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Storms will cause damage to palm trees close to edge of sea

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Difficult sampling technique due to e.g. uneven arrangement of palm tress

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Laboratory conditions may not relate to what happens in reality/real life situation

We assume that the species is evenly distributed throughout the area and that the placing of the quadrats is entirely random -

Movement of organisms

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Sampling taken within a small amount of time

2. ​Effect of Temperature / Conc. On Development of an Organism

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Safety:

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Tissue culture may provide good growing conditions for bacteria

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Possibility of an allergic reaction to the plant growth regulators / plant material

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Use of sharp instruments

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Preliminary Work:

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Practise proposed method to see if proposed method will work

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Determine appropriate dependent variable

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Check most suitable conditions for growth of plant tissue

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Select suitable timescale for measuring growth rates

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Check for other variables that need to be taken into account / controlled

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Check if the type of plant / tissue used is affected

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Check for (suitable) range of concentrations of plant growth regulator to be used

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Variables to be controlled are:

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Same age / size of organism

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Same parent organism

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Light intensity

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O2 concentration

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Mineral Concentration

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Water

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Food

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Time

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pH

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Method:

Dependent variable: E.g. percentage change in mass of plant tissue / no. of hatched shrimp / height for seedlings Independent variable: Concentration of plant growth regulator / temperature (at least 5 different ones) -

Take at least 5 repeats and calculate means

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Maybe give a control if appropriate

Specific descriptions of plant tissue culture provided (e.g. need to grow on nutrient gel, aseptic conditions, antibiotics in gel to prevent growth of microorganisms…) ​Same as for Totipotency and Plant Tissue Culture -

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Consideration of time period over which the growth will be measured

Limitations:

Difficult to control all variables affecting tissue growth + example e.g. exposure to bacteria -

Damage to plant tissue during preparation may affect growth

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Other limiting factor for plant tissue growth

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Need for more than one type of plant growth regulator for effective growth

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Any difficulties in the method you have proposed?

· An increase in environmental temperature causes the yield of some crops to decrease because less material is stored. As temperature increases, so does respiration / photosynthesis / metabolism which causes an increase in the GPP. Enzymes are more effective at higher temperatures below an optimum. Temperature has a greater effect on respiration than photosynthesis so NPP will be smaller as NPP = photosynthesis – respiration. Photosynthesis may be limited by another factor. 3. ​DNA Amplification Using PCR ·

Method:

A mixture is prepared containing: the DNA sample, DNA polymerases (with v. high optimal temperatures), DNA primers (short, single-stranded lengths of DNA that are complementary to those at the start of the STRs, they have fluorescent markers attached that aid the production of the final profile) and nucleotides

The mixture is the placed into a PCR machine where it undergoes the following cycle 1.

Sample is heated to 95 ºC à This separates the double helix into two strands

2.

Mixture is cooled to 55 ºC à Allows the primers to bind to the start of the STRs

3. Further heating to 70 ºC à DNA polymerases attach to the primers and extend them, replicating the STR sequence and the adjacent DNA 4. Cycle is repeated for about 25-30 times, which takes about 3 hours, to produce a mixture of different-length fragments unique to the individual

· The properties of the enzyme relevant to its biological activity in the amplification process: -

Enzyme is heat stable.

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Its optimum temperature is 70-80 ºC

It synthesises a new strand of DNA complementary to the template strand in one direction. A primer is needed to begin synthesis of the complementary strand.

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Effects of temperature on production of DNA:

In the context of denaturation of DNA / 90 to 95 ºC / step 1 à If temperature too low DNA strands will not separate. In the context of primer annealing / 40 to 70 ºC / step 2 à If temperature is too high less binding of primers will occur In the context of extension / 70 to 80 ºC / step 3 à If temperature is too low synthesis of new DNA strands would not be completed à If temperature is higher than 95 ºC, the enzyme will denature

· Collect and analyse samples from more than one individual of each species because: -

One individual does not represent the whole species

There will be genetic variation between individuals of the same species, testing more than one sample will control for these differences -

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Improves reliability of the data

Examine the DNA of 5 different genes because:

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Some genes might have little or no variation

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It will allow scientists to determine how closely they are related 4. Gel Electrophoresis

After using PCR, gel electrophoresis can be used to separate them according to their size and an image of the fragments produced · -

Method: Separating the fragments

1. The sample mixture is mixed with a coloured dye and placed carefully into wells at one end of agarose gel 2. The gel is immersed in a buffer solution in a tank and a potential difference is set up across it 3. DNA is –vely charged so will move towards the +ve electrode at the other end of the gel

4. Smaller fragments will move faster so mixture is separated out into a pattern of bands 5. The wells have a reference mixture of DNA fragments of known length to compare your samples to 1.

Seeing the fragments Fluorescent primers glow under UV light and allow a gel photo to be taken

2. DNA can be transferred from the gel to a nylon membrane by Southern blotting, which can be treated with a DNA probe. This binds to the bands and carries either a fluorescent or radioactive marker. Radioactive ones can be seen using autoradiography 3.

Coloured DNA probes can be added to gels to see the bands directly

Graphs can be plotted of the size of fragment against level of fluorescence (gives abundance of fragment) 5. Effect of Different Antibiotics on Bacteria

Same as Antimicrobial Properties of Plants

UNIT 5

6. ​Investigating Respiration

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Method: Place known mass of organism (maggots) into the respirometer.

Allow time for them to acclimatise to their surroundings and then move the drop of coloured liquid back to 0 on the scale using a syringe Start the stopwatch and note the position of the coloured liquid at regular intervals of 5 minutes. Subtract the final value from the first to give the overall distance moved. -

Use volume of oxygen uptake = π r2l (l = distance moved by liquid in tube)

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The volume divided by time will give the rate of respiration.

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Variables affecting respiration are:

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Type/source of seeds

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Mass/number of seeds

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Age of seeds

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Ph

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No. of organisms

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Temperature à Water baths between 20-40ºC

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Amount of soda lime

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Equilibration / acclimatisation e.g. Time left before measuring

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Moisture / humidity / water

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Time

· Yeast will respire faster using glucose because glucose is the starting point for glycolysis reactions in respiration; it is the first molecule to be phosphorylated. / Yeast will respire sucrose faster because it can be broken down into molecules of glucose and fructose; providing double the substrate for glycolysis / Yeast will respire sucrose more slowly because sucrose needs to be hydrolysed to glucose and fructose in order

to be used in glycolysis. / Rate of uptake of sugars differs: larger molecules may be taken up more slowly.

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Effects of:

No oxygen during investigation à No/less movement of the liquid in the respirometer. No/less change in volume/pressure of the gas. Aerobic respiration stops / Anaerobic respiration takes place. Anaerobic respiration produces no carbon dioxide. Increasing temperature à Will increase rate of respiration (as it is enzyme controlled) but also the volume of air in the apparatus -

Increasing air pressure à Will reduce the volume of air in apparatus 7. ​Effects of Exercise on Tidal Volume and Breathing Rate

· Method: This uses a spirometer. Adding air to the chamber makes the lid of the chamber rise in the water, and removing air makes it fall. Movements of the chamber are recorded using a kymograph (pen writing on a rotating drum). The volume of air the person inhales and exhales can be calculated from the distance the lid moves. The apparatus can be ​calibrated​ so that the movement of the lid corresponds to a given volume. -

The chart recorder can be set to move at a known speed.

A canister containing soda lime is inserted between the mouthpiece and the floating chamber. This absorbs the CO2 that the subject exhales. A ​disinfected mouthpiece​ is attached to the tube, with the tap positioned so that the mouthpiece is connected to the outside air. The subject to be tested puts a nose clip on ​(to ensure no breathing occurs via the nose),​ places the mouthpiece in their mouth and breathes the outside air until they are comfortable with breathing through the tube. Switch on the recording apparatus and at the end of an exhaled breath turn the tap so that the mouthpiece is connected to the spirometer chamber. The trace will move down as the person breathes in. After breathing normally the subject should take as

deep a breath as possible and then exhale as much air as possible before returning to normal breathing. Repeats could be for same student at same time each day for a week or with 10 different students (same age, gender, health, etc.)

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Variables to be controlled:

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Same person / age / gender / time of day

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Temperature

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Diet before testing…

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Standardise exercise

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Breathing must be measured over a set time (e.g. 5 minutes)

· How breathing is controlled by the nervous system in response to changing positions e.g. standing up and sitting down: More energy is needed when standing up. The sympathetic nerve increases heart rate. The ventilation centre in the medulla responds to chemoreceptors in the carotid that detect changes in levels of carbon dioxide in blood. Motor cortex. Nerve impulses go to muscles involved in breathing. 8. ​Investigating Habituation to a Stimulus ·

Ethical issues:

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Snails must be handled carefully so as not to harm or stress it

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Snails should be released into the wild after use

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Safety:

- Snail secretions may irritate skin or cause allergies or carry microbes à hands should be washed thoroughly before and after handling snails

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Variables that affect it are:

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Temperature

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Background Noise

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Humidity

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Light Intensity

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Species

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Age

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Gender

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Method:

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Dependent variable: Time taken to fully emerge

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Independent variable: Number of pokes

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Place snail on clean, firm surface

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Allow time until snail has fully emerged from shell and has acclimatised

With a moistened cotton wool bud, firmly but carefully touch the snail between the eye stalks, starting the stopwatch immediately -

Record the time taken for the snail to fully re-emerge

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Repeat the touch and timing for a total of 10 touches

· Outcome: As the number of stimuli increase, the time taken for the snail to re-emerge decreases.

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Limitations:

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Snails already handled before the experiment may not react in the same way

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Determining when a snail has fully emerged

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Lack of moisture may encourage snail to stay more in its shell

· An increase in temperature increases the rate of chemical reactions in the snail, allowing it to re-emerge more quickly. To control temperature use incubator/conditioner at constant temperature suitable for snail

· Calcium ion involvement in habituation: Repeated stimulation affects calcium channels. Fewer calcium ions enter the pre-synaptic membrane and so less neurotransmitter is released into the synaptic cleft. Less depolarisation of the post-synaptic membrane will occur and fewer sodium channels will open. No action potential will be generated and so no impulse sent, no response is observed.