Required practicals 4-6

Required Practical 4

Investigation into effect of a named variable on the permeability of cell-surface membranes

Describe a method to investigate the effect of a name variable on the permeability of cell membranes

• Cut equal sized cubes of plant tissue (beetroot e.g.) of same age/ type using a scalpel

• Rinse to remove pigment released during cutting or blot on paper town

• Add same number of cubes ti 5 different test tubes containing same volume of water

• Place each test tube in water baths of different temperatures (e.g. 10, 20, 30)

• Leave for same length of time

• Remove plant tissue and measure pigment release by measuring intensity of colour or concentration of surrounding solution semi-quantitively or quantitively

How can we semi-quantitively estimate pigment concentration in a solution?

• Use a known concentration of extract and distilled water to prepare a dilution series

• Compare results with these colour standards to estimate concentration

How can we quantitatively estimate pigment concentration in solution?

• Measure absorbance of light of known concentrations using a colorimeter

• Draw a calibration curve (absorbance on y axis and conc on x axis) and draw a line/ curve of best fit

• Read off sample absorbance value on curve to find associated concentration

Why is the beetroot washed before placing it in water?

• Wash off any pigment on surface

• To show that release is only due to the independent variable

Why is each test tube regularly shaken?

• Ensures all surfaces of cubes remain in contact with liquid

• To maintain a concentration gradient for diffusion

Why does the volume of water need to be controlled?

• Too much water would dilute the pigment so solution would appear lighter (more light would pass through colorimeter than expected)

• So ensures the results are comparable

How would you ensure the beetroot cylinders were kept at the same temperature throughout the experiment?

• Take reading at regular intervals using a digital thermometer/ temperature sensor

• Add hot/ cold water (corrective measure) if temperature has fluctuated

Describe the issues of comparison with a colour standard (semi-quantitively)

• Subjective

• Colour obtained may not match any of the colour standards

What would a high absorbance suggest about the cell membrane?

• It is more permeable as more pigment leaks out making surrounding surrounding solution more concentrated

How does temperature affect permeability of cell membranes?

• As temperature increases, permeability increases

  • Phospholipids gain kinetic energy so fluidity increases

  • Transport proteins denature at high temperatures as hydrogen/ ionic bonds break, which changes their tertiary structure

• At very low temperatures, permeability increases

  • Ice crystals can form which pierce the cell membrane and increase permeability

How does pH affect permeability of cell surface membranes?

• High or low pH increases permeability

  • Transport proteins denature as hydrogen/ ionic bonds break changing the tertiary structure

How do lipid soluble solvents like alcohol affect permeability?

• As concentration increases permeability increases

• As solvent may dissolve phospholipid bilayer, which would create gaps

Describe a suitable control for this investigation and why a control is important (if the variable was alcohol conc)

Distilled water and two beetroot discs to show that it was alcohol which caused the leakage of pigment

Why must the beetroot not be cooked?

Heating would damage the membrane/ denature proteins so less pigment would be present in tissue

REQUIRED PRACTICAL 5

Dissection of animal or plant gas exchange system of organ within such a system

Describe precautions that should be followed when performing a dissection

• Cover any cuts with a waterproof dressing

• When using a scalpel, cut away from body onto a hard surface and make sure the blade is sharp

• Keep the blade of the scalpel protected/ pointing fown

• Wear disposable gloves and disinfect hands/ wash with soap

• Disinfect surfaces/ equipment

• Safe disposal of organ: put any used gloves/ tissues/ organ in separate bag/bin to dispose if

• If poisonous chemicals/ toxins involved work in a well ventilated environment

What are ethical considerations when dissecting animals

• Morally wrong to kill animals just for dissection

• So should use animals for dissection that have already been killed for meat

How could you prepare a temporary mount of piece of plant tissue for observation with an optical microscope?

• Add a drop of water to glass side

• Obtain a thin section of specimen and place on slide

• Stain (e.g. with iodine to view starch)

• Lower coverslip at 45 degree angle using mounted needle without trapping air bubbles

What are the rules of a scientific drawing?

• Add magnification scale

• Add labels

• No sketching; only straight continuous lines

• No shading

• Parts drawn to the same scale/ relative size

REQUIRED PRACTICAL 6

Use of aseptic techniques to investigate the effect of antimicrobial substances on microbial growth

What are some examples of aseptic techniques that could be used?

• Wash hands with soap/ disinfectant surfaces

  • Kills microbes/ prevents contamination

• Sterilise pipette/ spreader/ boil agar medium

  • Kills microbes/ prevents contamination

• Flame neck of bottle of bacteria

  • Kills microbes/ prevents contamination

• Bunsen burner close

  • Upward current of air draws air-borne microbes away to prevent contamination

• Minimise opening of petri dish

  • Prevents entry of microbes (contamination)

Describe a method to investigate the effect of antimicrobial substances in microbial growth

• Prepare area using the above aseptic techniques

• Use a sterile pipette to transfer bacteria from broth to agar plate using aseptic techniques

• Use a sterile spreader to spread bacteria over agar plate

• Use sterile forceps to place same size discs that have been soaked in different concentrations of antimicrobials (for the same length of time) onto agar plate at equal distances apart

• Lightly tape lid onto plate (not fully sealed), invert and incubate at 25 degrees for 48 houra

• Measure diameter of inhibition zone around each disc and calculate area using πr²

Why is it important to maintain a pure culture of bacteria?

• Bacteria may outcompete other bacteria being investigated

• Or could be harmful/ pathogenic to humans

Why is the lid held with 2 pieces of tape and not sealed completely?

• Allows oxygen in to prevent growth of anaerobic bacteria

• Which is more likely to be harmful/ pathogenic to humans

What could be used as a control?

• A paper disc soaked in water (no microbial agent)

• To ensure it was the antimicrobial which prevented growth, not the paper disc

Why are Petri dishes incubated upside down?

• Condensation drips onto lid rather than surface of agar

How can zone of inhibitions be measured if the shape is irregular?

• Repeat readings in different positions and calculate a mean

Why is a higher antimicrobial concentration not used?

More bacteria killed so clear zones may overlap

Why should bacteria be incubated at 25 degrees or less in school/ labs?

Below human body temp so prevents growth of pathogens

Explain the presence and absence of clear zones

Clear zones

• Antimicrobial has diffused out of disc into agar, killing/ inhibiting growth of bacteria

  • Larger clear zones= more bacteria killed= more effective antimicrobial

No clear zones

• If an antibiotic was used, bacteria may be resistant or antibiotic may not be effective against that specific bacteria

How can data about the effect of antimicrobial substances be presented as a graph?

Categorical data

• Use a bar chart: x axis is type of antimicrobial and y axis is zone of inhibition

Continuous data

• Line graph joined by line of best fit: x axis is concentration of antibiotic and y axis is area of zone of inhibition