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AICE Biology
Lab Review:
Part 1
FYI
• 1 cm3 = 1 mL
• Always add 3 mL of biurets or
benedicts
• 2 mL of iodine should be used in
starch tests
B. Biological
Molecules (a) carry out tests for
reducing and non-reducing sugars (including semi-quantitative use of the Benedict’s test), the iodine in potassium iodide solution test for starch, the emulsion test for lipids and the
biuret test for proteins;
Benedict’s Test: Reducing & Non
Reducing Sugars, Round 1
RESULTS:
Aqua-blue = negative.
Green to Yellow to orange = positive. Note: to detect the simple sugar sucrose, you must do round 2 test
Left to right:
• Benedict's reagent (BnR),
• potato extract + BnR,
• onion extract + BnR,
• 5% glucose + BnR.
Testing for simple sugars: Benedict's Reagent, round 1
Procedure:
Add Benedicts reagent to
sample, place in waterbath
until just boiling.
Benedict’s Test: Reducing & Non
Reducing Sugars, Round 2
A Negative result in round one DOES NOT
mean an absence of carbohydrates!!
Sucrose is a non-reducing Sugar
& can only be detected by doing
round 2 of Benedict’s testing
(Acid Hydrolysis)
Procedure:
• Perform round 1 of Benedict’s Testing. Negative result indicates
either no carbohydrate OR Non-reducing Sugars (Sucrose or starch).
• How would you test for the presence of starch???
• Perform Round 2 of Benedict’s Testing to see if you have sucrose if
Starch test is Negative.
Sample Procedure for
Benedict’s Test for Non
Reducing Sugar
• In a test tube place 5 cm3 solution, add 3 cm3 Benedict's reagent to the solution in the test tube and place the tube in the boiling water bath for five minutes.
• Add 1 cm3 dilute hydrochloric acid to the solution solution in your test tube. Note the time and place in the water bath
• After 10+ minutes, remove the tube from the water bath & cool it under the tap. Neutralize the acid by adding solid sodium bicarbonate, a little at a time, until the addition of one portion produces no fizzing.
• With a dropping pipette place 3 cm3 Benedict's solution in test tube & return to the water bath and heat for five minutes.
• A color change indicates that there was Non reducing sugar present. How would you verify that your solution was sucrose & not starch???
Benedict’s Testing:
Sample Question 1
Solutions of four food substances are
tested for sugars. The table shows the
colours of the solutions after testing.
Which food is a non-reducing sugar?
Benedict’s Testing:
Sample Question 2
Four sugar solutions were tested with a standard Benedicts solution. The table shows the colour of the solutions after testing.
What is the best interpretation of the results?
Testing for Starch
IKI (Iodine in
Potassium Iodide)
RESULTS: Yellow-orange = negative.
Purple-black = positive.
Left to right:
• IKI only,
• starch solution,
• starch solution +
IKI.
Emulsion Testing
for Lipids Sample Procedure:
• Add 2cm3 fat or oil to a test tube containing 2cm3 of absolute ethanol. Dissolve the lipid by shaking vigorously. Add an equal amount of cold water.
• Observation: A cloudy white suspension.
Basis of test:
•Lipids are immiscible with water. Adding water to a solution of the lipid in alcohol results in emulsion of tiny droplets in the water which reflect light and give a white , opalescent appearance.
Testing for Polypeptides
(proteins)
Biuret’s Reagent
Left to right:
• Biuret's reagent (BrR),
• water + BrR,
• egg albumin solution,
• egg albumin solution+ BrR.
RESULTS:
Denim-blue = negative.
Lavender = positive.
Identify each sample
Food tests are carried out on four unknown chemicals. The chart below shows the results of each test.
Solution Benedict’s
Test
Acid hydrolysis then
Benedict’s
IKI Biuret Emulsion
A X + X X X
B X X X + X
C + X X + +
D X + + X X
(KEY: + = positive result , X = negative result)
C. Enzymes
(c) follow the time course
of an enzyme-
catalysed reaction by
measuring rates of
formation of products
(for example, using
catalase) or rates of
disappearance of
substrate (for
example, using
amylase)
Enzymes
(d) investigate and explain the effects of temperature, pH,
enzyme concentration and substrate concentration on the rate
of enzyme-catalysed reactions, and explain these effects;
Practice Question 1:
The curve X shows the activity
of an enzyme at 20oC. Curves
A to D show the effect of
different conditions on the
activity of the enzyme.
Which curve shows the effect of increasing the
temperature by 10o C and adding extra substrate?
Enzymes Practice Question 2
The graphs show the effects of temperature and pH on
enzyme activity.
Which statement explains the enzyme activity at the point shown?
A. At P, hydrogen bonds are formed between enzyme & substrate.
B. At Q, the kinetic energy of enzyme and substrate is highest.
C. At R, peptide bonds in the enzyme begin to break.
D. At S, the substrate is completely denatured.
Enzymes
Practice Question 3
The graph shows the effect of substrate concentration on
the rate of an enzyme-controlled reaction. The enzyme
concentration is constant.
Which statement about the graph is correct?
A. Between W and X, the number of
enzyme molecules is limiting.
B. Between X and Y, the number of
enzyme molecules is limiting.
C. Between X and Y, the number of
substrate molecules is limiting.
D. Between X and Y, the product
concentration remains the same.
Independent variable
• The factor whose values YOU decide
on
• You know this ahead of time
• Always on x-axis
• YOU change
Dependent Variable
• The variable NOT under YOUR control
• Depends on what happens with the
independent variable
• This is what you are
measuring/collecting data about
• You do not know these values until the
experiment is complete
Variables
• Continuous
– You can choose any value within the range you have
decide to use (for IV)
– Ex. How does temperature affect the rate of breakdown of
hydrogen peroxide?
• You need to decide on five set values within a temperature range
• Discontinuous
– There is a limited number of possible values
– Ex: Is the density of stomata on the lower surface of the
leaf greater than the density of stomata on the upper
surface of the leaf?
• There are only 2 possible “values” : upper or lower, no range
necessary
Determine Independent &
Dependent Variable
• Examples
– How does enzyme concentration affect
the rate of the reaction?
– How does temperature affect rate of the
reaction?
– How does concentration of solution affect
the percentage of onion cells that have
plasmolyzed?
Standardized/Controlled
Variables
• All other variables that may affect the
outcome of the experiment
• Must be kept constant and the same every
time you do the experiment
• Changing these variable would change the
result of your experiment
• You would not be able to determine affect of
the independent variable
• Know which ones need to be constant and
which do not matter (know your experiment)
Determining Independent
variables
• Determine appropriate range and intervals for your
experiment
• Range
– Spread of values from lowest to highest
– How to determine your range
• What is the concentration of the stock solution?
– This should be your highest/most concentrated value
– You will then make a range of solutions more dilute than the stock solution
• Interval
– “gap” between the values that you choose within your range
– Ex. 0, 0.2, 0.4, 0.6, 0.8, and 1.0%
– Interval is 0.2%
– You should always have at least FIVE values in your range
Dilutions
Simple Dilution
• Unit volume of liquid of
interest is combined with
an appropriate volume of
solvent liquid to achieve
desired concentration
– “1 to 5” dilution means:
• 1 unit of solute AND 4
units of solvent for a total
volume of 5 units
Serial Dilution
• A series of simple dilutions
which amplifies the dilution
factor quickly beginning with
a small quantity of material
• The solute for each step
comes from the previous
dilution
Measuring your Dependent Variable
• Look at the type of experiment
– Example: Testing the affects of enzyme concentration on
enzyme activity
• Options:
– Determine initial rate of reaction
• Take measurements very quickly to find out how much product is
formed/or how much substrate has disappeared in the first minute of
reaction
– Determine time for reaction to be complete
• Must wait until all substrate has been converted to product (could
take long time)
– Determine how long it takes reaction to reach an end-
point (clearly identifiable stage of reaction)
Measuring Color in Dependent Variables
• Color standards – Carry out macromolecule test on set of solutions
with KNOWN concentrations (you make these)
• Use excess of your indicator
– This produces a range of colors (and you know
the concentrations)
– Stand this in a test tube rack
– Now test your unknown sample
– Compare sample to your known concentrations
and determine the concentration of your sample
• Terms to use
– Simple words: red, blue, purple, green
– Qualify with “pale” or “dark”
– Use +, ++, +++ to show intensity (include a
key)
– State actual color…do not say “no change”
Recording Quantitative
Results
• Descriptions of what you see
• Use simple language
• Avoid terms that would be difficult to
understand (ex. yellowish-green)
– Should say “this tube is darker or lighter
green than tube 1”
• Never state “no change” say the color
– Example…if there was no reaction to
benedicts solution, you would state, “the
tube remained blue”
Tools to Measure
Dependent Variable
• Colorimeter
– Measures color changes
– Quantitative measurements of
color intensity in solution
– Good way to “improve
reliability” of experiment
– Uses cuvettes that contain
solution
– Deeper colors absorb more
light
– Important to choose suitable
color of light to shine through
(opposite of the color of
solution)
Tools to Measure
Dependent Variable
• Haemocytometer
– Counts cells in a given
volume (if it is a given area,
we can just use a grid in the
eye piece)
– It’s a slide with two sets of
ruled grids in center and
deep grooves on either slide
– Surface between grooves in
0.1 mm lower than the rest of
the slide
• Creates a “counting chamber”
Common independent or
standardized variables
• Temperature
– Control with warm water bath
• pH
– Use buffer solutions
Warm Water Bath
• Materials
– Large beaker of water
– Thermometer
– Ring stand
– Bunsen burner
– Wire gauge
– Thermometer clamp
• Measure temperature carefully
– Use thermometer in water
– Cannot touch bottom of beaker of water or the
side
– Read thermometer while it is in the water
– Allow test tubes in warm water bath to reach
same temp as water • Should measure actual temp of liquid in tubes
• Enzyme Experiments
– Bring enzyme & substrate solution to same temp
before adding to one another
– Keep both in separate tubes in same warm water
bath
Controlling pH
• Use buffer solutions
– Have a specific pH
– Maintains an even pH even if
reactions produces acid or base
– Add measured amount of buffer
to reacting mixture
• Use an indicator to measure pH
• Universal indicator= range from
0 to 14
****More reliable: pH meter
Controlling Biological
Samples
• Difficult
• Make sure all are identical
• Features to try and keep the same: – Age
– Storage conditions
– Genotype
– Sex
– Mass
– Volume
– Position in the organism from where sample was
taken
Other VARIABLES to control
• Light Intensity
– Vary distance of light source (light intensity is proportional to distance)
– Use plastic between plant and light source to prevent heat from
becoming a factor
• Wind speed
– Use a fan
– Use different distances
• Humidity
– Water content in air
– Use container of water near plant
• Increases humidity
– Cover plant with plastic bag
• Increases humidity
– Calcium chloride close to plant
• Absorbs water vapor
• Decreases humidity
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