bio core pracs

Core prac 1- factor affecting initial rate of reaction for enzyme

Method

1. Dilutions of 1% trypsin conch into 0.2%, 0.4%, 0.6% and 0.8

2. 2cm3 of trypsin and 2cm3 of water as reference cuvette to set up colorimeter

3. Measure 2cm3 of milk into cuvette, add 2cm3 of first trypsin conc, mix fast and measure w colorimeter every 15 s

4. Rise w water and repeat

Colorimeter

At aborbance, trypsin breaks down milk into colourless solution so higher absorbance as light passes easier

Why initial rate

Rate dec over time as substrate conc dec, we only know conc at the start so we can make a valid comparison

Scratch on cuvette- random or systemic error

Systemic, repeated use of scratched equipment despite calibrating the non scrratched one so higher results

Core pra 2 microscopes

Method

1. calibrate eyepiece graticule
2. cut transverse sections of plant stem (using a razor on white tile, wet) as thinly as possible. select the 2 thinnest
3. mount 1 section in water on a microscope slide. lower the cover slip down carefully onto the slide, with no air bubbles
4. add toliuidine blue O stain to the other + leave for 3 mins, then mount in water on another microscope slide + cover the slip
5. place under microscope + set objective lens on lowest magnification
6. use coarse adjustment knob to move the lens down
7. use fine adjustment knob to re-adjust,et focus
8.observe + draw ste,
9. measure the sze of the stem diameter + vascular bundle using the calibrated eyepiece graticule

How to calibrate

by placing both on the stage + lining up the divisions of the stage micrometer + the divisions of the eye piece graticule to calculate the length of 1 of the divisions

Risks

Stain is harmful if swallowed- gives and eye protection

Razor blades can cause cuts , cut stems away from fingers

Core prac 3

Risks

Hydrochloric acid- irritant, eye protection and gloves

Acetic orcein- harmful- gloves,fume cupboard

Method

1. Heat HCl

2. Leave roots in acid

3. Cut root, put in stain, heat to help staining

4. Maceration, coverslip, squash

5. High power mag

Why is the root tip heated with acid

Helps break down the pectates which hold the cell wall together

Why maceration

Separates and spreads out the cells so they are one thin layer and the chromosomes can clearly be seen

Core prac 4- sucrose conc and pollen tube growth

1. Make up solutions of sucrose between 0.2-2 moldm-3
2. Collect a petri dish for each conc and label
3. place filter paper in each dish, moisten paper with water
4. Collect one microscope slide per conc without touching centre
5. Place few drops of sucrose plus mineral salt medium in central cavity of each slide
6. Collect a flower that has mature anthers and is shedding pollen
7. Gently, rub point on needle over anthers so pollen falls on slide, do not add coverslip
8. repeat for each conc using same flower
9. note time at which pollen was added and place slides in petri dishes
10. use microscope at x100 magnification and a calibrated eyepiece graticule to observe slides, doing it quickly
11. Once pollen tubes have started to grow, measure length of four or five tubes at three min intervals for about 30 mins

Why is a Petri dish used

To prevent slides from drying out

Why is there no coverslip

Prevents anoxic conditions preventing respiration of pollen cells

Why are observations made quickly

Prev drying out

Sources of error

-flowers must be from same plant to ensure genetic consistency
-difficult to accurately measure length of pollen tube
-pollen must be from a ripe flower to ensure germination

Risks

H: needle, glassware
R: breakages, stick injuries
S: take care

H: pollen
R: allergy
S: take appropriate precautions

You were instructed to use pollen from only one flower for all the concentrations. Comment on
any advantages and disadvantages of this approach in terms of the validity and reliability of
results

Advantage: the use of pollen from the same flower improves the validity of the results because
the maturity of the flower is controlled so any differences in results are more likely to be a result of the differences in sucrose conc
Disadvantage: the results may not be reliable. Different results may arise if the experiment is
repeated because of differences between flowers. Flowers may differ slightly in maturity or due to genetic differences. Ideally, the investigation would be repeated with a large number of flowers and a mean would be taken

Role of pollen tube

The pollen tube carries the male gametes to the ovule for double fertilisation

Suggest what might cause pollen tubes to grow in the correct direction in the style.

The pollen tube grows in the correct direction due to chemotropism (chemical attraction), chemical attractants released from the synergies, and the physical structure of the style

Core prac 5- temp on beetroot membrane permeability

Method

1. Using cork borer and knife, cut pieces of beetroot into 1cm length cylinders
2. Place in distilled water overnight to remove any dye released on preparation, wash and blot dry
3. Place 8 boiling tubes of distilled water into 8 water baths of different temperatures, add a piece of beetroot to each and leave for 30 mins
4. Remove beetroot and shake tubes to disperse dye
5. Set colorimeter to % absorbance on blue/green filter, calibrate using distilled water in a cuvette first then add 2cm3 of beetroot solution from first temp to a new cuvette
6. Place into colorimeter to read % absorbance
7. Repeat

Control variables and how

● the volume of bathing water in each tube (10 cm3)
● the surface area and volume of the beetroot cylinders (dependent on size of cork borer; 1 or 2 cm in length)
● the equilibration time (5 minutes)
● the soaking time for the cylinders (15 minutes)
● the volume of coloured liquid in the cuvettes (e.g. 4 cm3)
● the colorimeter filter/wavelength used (blue/green)
● the part of the beetroot the core was taken from (e.g. the centre)
● the age, variety and storage time of the beetroot (the same beetroot or beetroots from the same batch may have been used).

Saftey

H: water baths temp above 50
R: burns
S: take care, allow steam to escape away from face

H: sharp items such as cork borer and knife
R: cuts
S: be careful

Evaluation issues

-beetroot may have skin on, affecting surface area
-difficulty in maintaining temp
-accurate reading of colorimeter
-accurate size of beetroot
-from different parts of root
-ensuring same amount of time a different temps

Suggest why the tubes were placed in the water baths for 5 minutes before the cylinders were added.

The temperature must be equilibrated to ensure the tubes contain water at the correct temperature. This allows confidence that the effect of the correct temperature is being assessed

Why were the beetroot cylinders washed with distilled water and dried before the experiment began?

to remove excess surface pigment from the cut cells at the edge. This excess pigment would distort the transmission readings, giving inaccurate results

Use the trend line of your graph to describe the effect of temperature on the percentage transmission between 0 °C and 70 °C.

The percentage transmission decreases as the temperature rises. Initially there is little increase, but at around 40-60 °C the percentage transmission decreases sharply. At higher temperatures the rate of decrease usually levels out.

Explain your results in detail in terms of what is happening to the beetroot membrane.

lower temp: the tonoplast and plasmalemma are intact and betalain molecules are too large to pass through these membranes so light transmission remains high.
higher temp: greater the kinetic energy, faster movement and diffusion of pigment molecules. Greater kinetic energy also causes phospholipids of the membrane to become more fluid and bonds between the fatty acid tails can begin to separate so that some pigment molecules can pass through. Therefore more pigment passes through the membrane, decreasing the amount of light that can pass through the liquid (percentage transmission).
The point of sudden increase in percentage transmission occurs when proteins in the membrane begin to lose their tertiary structure. At higher temperatures, the protein molecules in the membrane become completely denatured and the membrane develops gaps through which the pigment can flood out.
Eventually, the change in transmission levels out as the concentration of pigment is the same inside and outside

Core prac 7- water potential of epidermal cells

Method

1. Take 5 thin sections of red part of red onion (1cm2)
2. Label 5 watch glasses and add 5 different concs of sucrose solution
3. Add a piece of tissue to each glass and leave for 20 mins
4. Put a drop of each solution on slide
5. Remove each tissue piece using forceps and float each the matching slice
6. Cover each section with coverslip and observe
7. Count 25 cells and record how many show plasmolysis, record findings in table

Why thin sections

Easy to view under microscope

Evaluation issues

-difficult to obtain a single layer of red stained cells
-difficult to accurately measure just 25 cells by looking down the microscope
-the pigment varies between different parts of the onion

Could choose your 25 cells

Some cells look plasmolysed- subjective

Membrane damage when cut

Incipient plasmolysis

When 50% of tissue sample is plasmolysed

Water potential is described by the equation ψ = P + π. At the point of incipient plasmolysis the
cell membrane is just beginning to peel away and exerts no pressure on the cell wall, so P = 0.
Therefore ψ must equal π. There is no net movement of water by osmosis at this point.

Core prac 7- locust dissection

Method

1. Put live locust inside jair, look for spiracles on the thoracic and abdoninal segments using a hard lens
2. Tilt locust a little to look for rhythmic ventilation movements of the underside of abdomen
3. Take dead locust, view it from side using a dissedting microscope
4. Pin locust down. Cut off wings and remove exoskeleton by cutting along the side, then across the head
5. Flood with water to see tracheae
6. Tissue can be stained with methylene blue and viewed under a microscope

Ethical issues

- working with living organism so minimise harm
- all dissection should be purely for educational purposes

Use of locust which has a high population so wont go extinct, killed in a way which prevents suffering, can use large groups

Explain role of Spirals of thickened chitin in tracheae

The chitin spirals support the tracheae. They hold the tracheae open if they are squashed as the insect moves

Explain role of Ventilation movements of the abdomen

Ventilation movements actively pump air into the tracheal system and increase the supply of oxygen to very active tissues. Expansion of the abdomen increases the volume decreases the pressure inside the body so pressure in the tracheae becomes lower than atmospheric pressure, drawing air in through open spiracles. Compression of the abdomen decreases the volume and increases the pressure inside the body so that air moves out of the tracheae through open spiracles.

Explain role of air sacs

Air sacs act as air reservoirs or bellows. They increase the volume of air moved through the respiratory system. They have flexible walls so that changes in pressure caused by ventilating movements of the abdomen (or the thorax when in flight) inflate and deflate them.

Some insects have spiracles that have sphincters, allowing them to be opened or closed. Suggest two advantages of the ability to close and open spiracles.

Opening and closing of the spiracles allow the rate of gas exchange to be controlled. But water vapour also diffuses out of spiracles, so sphincters allow spiracles to be kept closed as much as possible to minimise the amount of water lost

Core prac 8

Method

1. set up potometer

2. clamp capillary tube into stand +p place bottom in the beaker of water

3. smear petroleum around the join to make airtight

4. leave for 5 mins to allow bubble to be drawn up

5. time length of time the bubble move a certain distance along tube

or

measure movement of bubble along tube in a certain length of time

6. convert measurement into rate of transpiration

7. repeat experiment + change abiotic variable

Why is it important to have an airtight seal

Without an airtight seal, water will nit be drawn up the capillary tube and in air bubble movement will be possible. Water movement in the capillary tube and in the xylem relies on cohesive forces between water molecules, air bubbles prevent the cohesive forces and stop the upward tension being transmitted through the entire water column

Limitations of procedure

Hard to control size of shoot, number of leaves, total surface area, leaves not fully dry

Factors reduce accuracy of equipment such as difficulty in making a seal between the shoot and apparatus

Effects:

Transpiration rate increases is a larger surface area is used or if there are more leaves present, wet leaves reduce diffusion gradient

Lack of airtight seal may stop transpiration- lower value for water uptake rate

How to reduce limitations

Shoots should be same size w same number of leaves. Leaf area should be measured so that water loss per unit area can be calculated

The seal must be airtight, this may be achieved by using a flexible material and adding a sealant, repeat gives reliability and mean