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