Practicals

Question 1

1. describe a method to investigate the effect of temperature on the rate of enzyme (trypsin) activity

Answer 1

1. make a control sample of just milk solution
2. add milk solution to 5 test tubes labelled with 20C, 30C, 40C, 50C & 60C
3. in separate test tubes at 2cm^3 trypsin solution & 2cm^3 pH7 buffer solution
4. put test tubes containing milk solution & trypsin with buffer into a water bath at 20C for 5 mins
5. repeat for all temperatures
6. add trypsin & buffer solution into the test tube of milk solution, place back into water bath & time for 4 minutes using a stopclock
7. after 4 mins, put contents of test tube into a cuvette into a colorimeter (calibrate colorimeter) & measure absorbance immediately
   (alternatively time how long it takes for solution to go colourless)
8. plot graph of temperature vs absorbance
9. the smaller the absorbance, the greater the rate of enzyme activity

(Repeat 5 times at each temp., calculate mean time taken & rate by 1/time & plot mean)

Question 2

2. describe a method to use an optical microscope to identify the stages of mitosis in root tip squashes & to calculate mitotic index

Answer 2

1. heat 1 mol of HCl in 60C hot water bath
2. cut root sample using a scalpel, place in HCl & leave in hot water bath for 5 minutes
3. wash roots in cold distilled water & cut the tips of the roots with a scalpel
4. place into acetic orcein stain & leave in water bath for 5 mins
5. place stained root tip on slide
6. place cover slip on top of root tip & squash them down
7. put the prepared slide under the microscope & set objective lens to lowest magnification
8. use the coarse & fine adjustment knob to focus the image
9. on highest magnification objective lens (X40), count the number of cells in mitosis & the total number of cells
10. make a biological drawing
11. mitotic index = # cells with visible chromosomes/total # cells

Question 3

3. describe a method to identify the water potential of potato tuber cells by a dilution series

Answer 3

1. make a series of dilutions with distilled water of 1M sucrose (or salt) solution at 0.0M, 0.2M, 0.4M, 0.6M, 0.8M & 1.0M sucrose
2. measure 10cm^3 of each dilution into separate test tubes
3. use a ruler & scalpel to cut out 6 potato chips of equal dimensions. blot with paper towel to remove excess water (do not squeeze)
4. weigh each potato chip using a mass balance
5. place 1 potato chip in each test tube & leave for 20 mins
6. remove each potato chip, blot dry with paper towel & reweigh them
7. calculate the % change in mass for each potato chip
8. plot graph of conc vs % change in mass

Question 4

4. describe a method to investigate the effect of temperature on the permeability of cell surface membranes (beetroot leakage)

Answer 4

1. cut beetroot into 10 equal dimension cubes
2. label 10 test tubes with relevant temperature (20-80) & put 5cm^3 distilled water in each
3. put each test tube in a thermostatically-controlled water bath at the given temperature
4. put 5 beetroot cubes into the water bath for 1 minute then put in labelled test tube for 20mins
5. after 20mins, pour coloured water into a cuvette & into a colorimeter
6. set colorimeter to blue filter, calibrate with distilled water, then measure absorbance
7. plot graph of temperature vs absorbance
8. higher absorbance = higher pigment conc. = higher membrane permeability

Can use pH buffer

Question 5

what must be done before using a colorimeter?

Answer 5

calibrate colorimeter with distilled water
= zero it

Question 6

5. describe precautions when dissecting e.g. a mammalian heart & lungs

Answer 6

disinfect surfaces before & after dissection
wear gloves
observe outside of heart & lungs before dissection
use scalpel & make sharp, precise cuts away from fingers

Question 7

6a. describe a method to investigate the effect of antimicrobial substances on microbial growth

Answer 7

1. use aseptic technique (describe throughout)
2. use a sterile pipette & flame neck of bottle to remove some E.coli broth
3. lift lid of petri dish facing the Bunsen flame & place a few drops of E.coli on the agar plate
4. flame a glass spreader, let it cool, then spread bacteria evenly on surface of agar
5. use sterile forceps to place filter paper discs soaked in antimicrobial solution onto the centre of the agar plate
6. secure the lid of the petri dish with 2 pieces of tape & put upside down in incubator @ 25C for 48 hrs
7. after incubation, measure the diamater & calculate the area of the zone of inhibition for each antibiotic using A=πr^2

Question 8

describe & explain examples of aseptic technique

Answer 8

wash hands with soap & disinfect surfaces
use sterile pipette
flame the neck of the bottle, glass spreader & forceps in a (roaring) Bunsen flame
lift the lid of the petri dish towards the Bunsen safety flame, which creates convection current/upwards air movement so microogranisms moved away
discard apparatus in a beaker of disinfectant

Question 9

6b. describe a method to investigate the effects of antimicrobial substances on the growth of bacterial populations in a broth culture

Answer 9

1. use aseptic technique (describe throughout)
2. use a sterile pipette & flame neck of bottle to remove some M. luteus broth & place 1cm^3 in the first bottle of disinfectant
3. remove 1cm^3 from first bottle & place in 2nd bottle & repeat for all bottles, removing 1cm^3 from final bottle & discarding it
4. for each dilution take 0.1cm^3 with a sterile pipette
5. lift lid of petri dish towards Bunsen flame & place dilution solution on agar plate
6. flame a glass spreader, let it cool, & spread dilution evenly over the surface of the agar
7. secure the lid of the petri dish with 2 pieces of tape & put upside down in incubator @ 25C for 48 hrs
8. after incubation, count individual colonies of bacteria, X by dilution factor X 10 to calculate an estimation for # of bacterial cells in original sample per mL

Question 10

9. describe a method to investigate the effect of temperature on the rate of respiration of cultures of single-celled organisms

Answer 10

1. set up a water bath at 35C
2. add 5cm3 of the yeast & glucose solution to a test tube & leave it until the contents reach 35C
3. add 2cm3 methylene blue to each test tube & start the timer. shake the tube for 10 seconds then put back into water bath
4. record how long it takes for the methylene blue to turn colourless for each test tube
5. repeat the experiment at 25C, 40C, 45C & 55C, 60C
6. find the mean mean time for each temperature & use it to calculate the average rate of respiration: rate = 1/time
7. plot a graph of temperature against rate of respiration

Question 11

10. describe a method to investigate the effect of an environmental variable on the movement of an animal using a choice chamber

Answer 11

1. set up choice chamber with 4 quadrants: dark & dry, dark & damp, light & dry, light & damp
2. use dark paper to block light on one half & wet paper towel to make damp areas. use anhydrous calcium chloride to make dry areas.
3. place 10 woodlice in centre of choice chamber & leave them for 10 mins
4. after 10 mins, record how many woodlice are in each quadrant
5. repeat by moving woodlice back into centre of choice chamber

Question 12

10. describe a method to investigate the effect of an environmental variable on the movement of an animal using a maze (did in class)

Answer 12

1. place a maggot at the start of the maze
2. record whether the maggot turnes left or right when it reaches a junction
3. remove the maggot & wipe to inside of the maze with cotton wool
4. repeat for 20 maggots
5. to test for turn alteration, place a maggot at point before a right turn
6. record whether the maggot turns left or right after turning right
7. repeat for 20 maggots, using a new maggot each time & wiping the maze in b/w tests

thigmotaxis

Question 13

how are the results of the maggot/woodlice experiment analysed?

Answer 13

Chi squared test - determines whether the observed result differ from the expected results
X2 = ∑(o-e)^2 / e
if the value exceeds the critical value at the 0.05 significance level, reject H0 –> statistically significant

Question 14

why must a new maggot be used each time?

Answer 14

to avoid the maggot learning which way to turn

Question 15

why is the maze wiped down each time?

Answer 15

to prevent chemotaxis - following the trail of the previous maggot

Question 16

describe a method to produce a calibration curve using a colorimeter

Answer 16

use distilled water & the sample to create a dilution series
use a known volume of solution
measure absorbance/transmission of the solutions of a known concentration using a colorimeter
plot graph of concentration of solution against absorbance/transmission

Question 17

11. describe a method to produce a dilution series of a glucose solution & use colorimetric techniques to make a calibration curve to identify concentration of unknown ‘urine’ sample

Answer 17

1. create a dilution series of glucose solutions using distilled water of conc. 1-10mmoldm-3
2. place 2cm3 of each unknown sample into separate boiling tubes & 2cm3 benedict’s solution to each
3. put boiling tubes in water bath & heat
4. measure the absorbance of each known sample using the colorimeter
5. plot graph of the known concentration of solution against absorbance/transmission = calibration curve
6. measure the absorbance of the unknown samples using the colorimeter & read off the concentration from the calibration curve

Question 18

12. describe a method to investigate the effect of a named environmental factor on the distribution of a given species

Answer 18

1. use 2 tape measures to split the study area into a grid with coordinates
2. use a random number generator to randomly select a set of coordinates
3. place the (bottom left of) 0.25m2 quadrat at the coordinates & count the # of individuals in the quadrat (or % cover by counting how many of the 100 squares in quadrat contain the given species)
4. repeat at least 20 times
5. calculate a mean, find area of field & multiply together to estimate population size
   NB if investigating light intensity, use photometer can be take a reading for light intensity

Question 19

/mm2 using a microscope

Question 20

what is the purpose of a control experiment?

Answer 20

shows the effect is due to independent variable alone
to allow comparison

Question 21

describe how to draw a labelled biological drawing/diagram

Answer 21

no shading
use single, continuous lines
include scale/magnification
ensure drawing is large enough for details & labels
sharp pencil