1.
2 Monosaccharides
AQA Biology Method sheet
Serial dilution: finding the minimum sensitivity
of Benedict’s and Clinistix tests
Specification references
• 7.1
• AT f
• AT c
Learning objectives
After completing the practical you should be able to:
• carry out a serial dilution of glucose solution
• use the diluted solutions to test the sensitivity of two tests for sugars: the
Benedict’s test for reducing sugars and the Clinistix test for glucose.
Background
In serial dilution, you will dilute a 10 % glucose solution through a series of dilutions,
going down to a 0.001 % glucose solution. This is faster and more accurate than
making each solution of the series from scratch. Note that small volumes of very
dilute solutions require very small masses of solutes that are too light for laboratory
balances.
You will then use these solutions to test the sensitivity of two tests for sugars: the
test for reducing sugars using Benedict’s reagent and the Clinistix test for glucose.
This investigation is split into three parts. You will need to make observations and
record results from each section before you can move onto the next. Keep your
equipment set up until you have completed the comparison between the two tests.
Safety
• Wear eye protection – there is some risk of hot liquids spitting when heating test
tubes.
• Benedict’s reagent: LOW HAZARD.
• Take care with glassware.
Equipment and materials
• eye protection • waste beaker
• labelling equipment • paper towel
• 10 × test tubes, bungs for test tubes • heating equipment: Bunsen burner,
• test tube holder bench mat, tripod, gauze, 400 cm3
beaker half-filled with tap water (to
• 10 cm3 pipette and pipette filler act as a water bath), thermometer
• 1 cm3 pipette and pipette filler • distilled water wash bottle
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1.2 Monosaccharides
AQA Biology Method sheet
• white tile • Clinistix test strips
Method
Step 1: Making serial tenfold dilutions of a 10 % glucose solution
1 Label four test tubes, so that you can keep track of which dilution is in which test
tube.
2 Add 9.0 cm3 of distilled water to each of the four labelled test tubes.
3 Add 1.0 cm3 of the 10 % glucose solution to the first test tube. Put a clean bung
into the neck of the test tube. Hold it in tightly and shake the test tube carefully
10 times to get an even mixture. This process achieves a tenfold dilution.
4 Using a clean pipette and a clean bung, repeat steps 2–3 for the three remaining
test tubes, on each occasion transferring 1.0 cm3 of your most recently diluted
glucose solution into the next test tube that contains 9.0 cm3 of distilled water.
Step 2: Devising a standardised test
For you to determine the minimum sensitivity of the test for reducing sugars with
Benedict’s reagent, you need to conduct the test in precisely controlled conditions,
and you need a control. You should already be familiar with the test but there is a
recap of the main procedure below.
A reminder of the ‘rough’ qualitative test for reducing sugars
1 Dispense about 2 cm3 of the test solution into a test tube.
2 Add 10 drops of Benedict’s reagent.
3 Heat to above 70 °C in a water bath for up to two minutes.
4 Observe the colours formed in the test tube against a white background,
e.g. a white tile.
Pointers towards devising a standardised test:
In a trial, the mass of Benedict’s reagent that was dispensed by two methods was
compared.
Method of dispensing Mass of the dispensed Benedict’s reagent / g
the Benedict’s reagent
Individual values Mean
10 drops from a dropper
0.34 0.37 0.39 0.35 0.39 0.37 (to 2 d.p.)
pipette
0.5 cm3 from a 1 cm3
0.58 0.59 0.58 0.59 0.59 0.59 (to 2 d.p.)
glass pipette
© Oxford University Press 2014 www.oxfordsecondary.co.uk/acknowledgements
This resource sheet may have been changed from the original 2
1.2 Monosaccharides
AQA Biology Method sheet
Questions
1 From the data in the table, suggest an advantage of using measured volumes
from a 1 cm3 glass pipette rather than 10 drops from a dropper pipette in a
standardised Benedict’s test.
2 List other variables that you think should be kept constant for a standardised test
for reducing sugars using Benedict’s reagent.
3 Suggest a control for the experiment to find the minimum sensitivity of the test
for reducing sugars using Benedict’s reagent.
4 Write down your method to determine the minimum sensitivity of the Benedict’s
test for reducing sugars using Benedict’s reagent (and check it with your
supervisor).
5 Use your standardised method to carry out the Benedict’s test on suitable
volumes of the glucose solutions that you have made up in the dilution series.
Record your results in the results table below.
Hint: consider whether all of the tests can be carried out in the same water bath at
the same time if there is staggered adding and removing of the test tubes.
Results
Table 1 Observations and deductions from tests for reducing sugars using
Benedict’s reagent on dilutions of 10 % glucose
Glucose
Observations Deductions
concentration / %
10
1.0
0.1
0.01
0.001
Conclusion for the test for reducing sugars using Benedict’s reagent
What was the lowest glucose concentration where reducing sugars were shown
to be present (when compared with the control and against a white background)?
Comparing the minimum sensitivity of the test using Clinistix and the test
using Benedict’s reagent
Now test a fresh sample of the least concentrated glucose solution that gave a
positive reaction with the test using Benedict’s reagent with the Clinistix test strip.
© Oxford University Press 2014 www.oxfordsecondary.co.uk/acknowledgements
This resource sheet may have been changed from the original 3
1.2 Monosaccharides
AQA Biology Method sheet
(If appropriate, test more dilute glucose solutions.)
Clinistix contains glucose oxidase and peroxidase in the coloured pad and is used
to detect glucose in urine as a preliminary test for diabetes.
Discussion
1 Was Clinistix able to detect the same low concentration of glucose as the test
using Benedict’s reagent? Could it detect lower values?
2 Suggest:
a Why Clinistix would not show the presence of 1 % fructose but the test using
Benedict’s reagent would.
b Why Clinistix would fail to show the presence of reducing sugars in a 1 %
glucose solution if the test strip had been accidentally dipped into some
hot water.
© Oxford University Press 2014 www.oxfordsecondary.co.uk/acknowledgements
This resource sheet may have been changed from the original 4
