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enzymes explained

Starch agar experiment A





Introduction

The starch agar used in this experiment consists of a mixture of soluble starch (0.5%) and agar (2.5%) to set it into jelly. This agar is not being used to culture bacteria or other micro-organisms, but instead to show how the enzyme amylase may break down its substrate starch to different extents depending on its concentration.


The starch agar was mixed, heated to sterilise it, then whilst still molten it was poured in a thick layer into sterile Petri dishes. After it set, it had 6 holes cut in it using a heat-sterilised cork borer, and the discs were removed aseptically, i.e. so as to reduce the likelihood of bacteria getting onto the agar.


You will be placing different concentrations of amylase into the holes or wells left by this process, allowing it to diffuse out sideways into contact with the starch, and afterwards you will be using the iodine test to see what has happened to the starch in the agar.

Preparation of a 10-fold dilution series

1) Place 5 test-tubes in a rack, and label them 1-5.

2) Using a measuring cylinder, put 9 ml of distilled water into tubes 2, 3, 4 and 5.

3) Pour a small amount (say 3ml) of 1% amylase solution into tube 1.

4) Using a fresh pipette, transfer 1 ml of the contents of tube 1 into tube 2 and mix by shaking.

Return the pipette to tube 1.

5) Using a fresh pipette, transfer 1 ml of the contents of tube 2 into tube 3 and mix by shaking.

Return the pipette to tube 2.

6) Using a fresh pipette, transfer 1 ml of the contents of tube 3 into tube 4 and mix by shaking.

Return the pipette to tube 3.

7) Using a fresh pipette, transfer 1 ml of the contents of tube 4 into tube 5 and mix by shaking.

Return the pipette to tube 4. Get another pipette for tube 5.

What have you done in each stage 4-7?

> dilution x10, x100, x1000, x10 000

Transfer of enzyme preparations to wells in starch agar plates


n.b. Only open the lid of the Petri dish as far as necessary, and close it immediately you have transferred the enzyme. Take care to keep the Petri dish level on the bench, and not to jog or spill it.

8) Using the template provided overleaf, put a mark on the edge of the (base of the) Petri dish. This is to locate the well numbers 1-6.

9) Using each tube's original pipette, transfer 2 drops of the contents of each tube to wells 1-5. Leave well 6 empty.

Label the lid of the Petri dish and leave it on the bench. It will be placed into the incubator at 36 C.

The Petri dish will be examined later - either at the end of the lesson or later - either the next day (come in at lunchtime to see) - or next week 10) After the appropriate time, flood the top of the agar with a few drops of iodine solution and angle it to spread over the whole surface.

Note the areas which stain and those which do not react to iodine. Draw outlines on the template to show the size of any clear zones.

Registration mark

v
stapet
Results taken > ... hours after start of experiment.

You started the dilution series with 1% amylase, i.e. 1 g dissolved in water and made up to 100 ml.
A small sample of this was placed into well number 1, and the same sized samples were put in the others.

In the space above, write the concentration of amylase in each well.

What does the main darkly staining area represent?

> starch

What do the non-staining areas represent?

> no starch

As accurately as you can, measure the diameter of the zones round the wells and record them under the well number above.

What is the significance of the different sized zones round each well?

>different amounts of amylase diffuse out, so different amounts of starch disappear

Why was well 6 left empty?

>as a control to check the effect of no amylase/to see that starch agar reacted with iodine

What do you conclude from this experiment?

>The more amylase there is, the greater the zone of starch breakdown

Starch agar experiment B

Introduction

The starch agar used in this experiment consists of a mixture of soluble starch (0.5%) and agar (2.5%) to set it into a jelly, together with a small amount of universal indicator. This agar is not being used to culture bacteria or other micro-organisms, but instead to show how the enzyme amylase may break down its substrate to different extents depending on the conditions in the surrounding medium, notably pH - the measure of acidity or alkalinity.

The starch agar was mixed, heated to sterilise it, then whilst still molten it was poured in a thick layer into sterile Petri dishes. After it set, it had 6 holes cut in it using a heat-sterilised cork borer, and the discs were removed aseptically, i.e. so as to reduce the likelihood of bacteria getting onto the agar.


You will be placing enzyme preparations with different pHs into the holes or wells left by this process, allowing it to diffuse out sideways into contact with the starch and afterwards you will be using the iodine test to see what has happened to the starch in the agar.

Preparation of a series of different pHs

CARE! Wear eye protection when handling acids and alkalies.

Do not use pipettes for any other purpose.

1) Place 6 test-tubes in a rack, and label them 1-6.

2) Put 10 ml of 0.1% amylase into tubes 1-6.

3) Using one pipette, add 2 ml acid to tube 1 THEN ..

4) Add 1 ml acid to tube 2 THEN ..

5) Add 0.5 ml acid to tube 3 THEN PLACE PIPETTE IN WASH.

6) Using another pipette, add 0.5 ml alkali to tube 4 THEN ...

7) Add 1 ml alkali to tube 5 THEN ..

8) Add 2 ml alkali to tube 6 THEN PLACE PIPETTE IN WASH.

9) Insert a fresh pipette into each of tubes 1-6 and mix the contents. Remove a drop from each tube and take the pH with the paper provided.

Record these values in the spaces overleaf.

Transfer of enzyme preparations to wells in starch agar plates


n.b. Only open the lid of the Petri dish as far as necessary, and close it immediately you have transferred the enzyme. Take care to keep the Petri dish level on the bench, and not to jog or spill it.

10) Using the template provided overleaf, put a mark on the edge of the (base of the) Petri dish. This is to locate the well numbers 1-6.

11) Using each tube's original pipette, transfer 2 drops of the contents of each tube to wells 1-6.

Label the lid of the Petri dish and leave it on the bench. It will be placed into the incubator at 36 C.

The Petri dish will be examined later - either at the end of the lesson or later - either the next day (come in at lunchtime to see) - or next week.


After the appropriate time, note coloured areas due to acid or alkali you have added - mark these in outline on the template - and see how these relate to the pH you measured before.

Then flood the top of the agar with a few drops of iodine solution and angle it to spread over the whole surface.

Note the areas which stain and those which do not react to iodine. Draw outlines on the template to show the size of any clear zones.

Registration mark
stbpet

Results taken ... hours after start of experiment.

What does the main darkly staining area represent?

>agar containing starch

What do the non-staining areas represent?

>agar containing no starch - because it has been changed/digested by amylase into a simple sugar (maltose) - which does not react with iodine

What is the significance of the different sized zones round each well?

>larger zones - more starch digested - nearer to optimum pH?

What do you conclude from this experiment?

> In order to digest starch, amylase needs a pH near to neutral/slightly acidic





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