January : Factors Affecting the Rate of Photosynthesis

We had an oxygenating aquarium plant (Cabomba), lamps, a supply of hot and cold water, and thermometers - and various other things which were handy.  Our mission: to investigate factors affecting the rate of photosynthesis.  We followed the classic Nuffield Practical Science method to investigate the rate of photosynthesis.

Our pondweed cuttings were inserted into a test tube of water, cut stem facing upwards.  The test tube was placed in a beaker of water.  We moved the beaker towards the lamp until bubbles started to appear from the cut end of the stem.  We counted the number of bubbles arising in a minute, then tried varying one condition to see how it affected the rate of photosynthesis.

Some groups measured distance from the lamp to the pondweed beaker.

Some tried varying the water temperature:

Adjusting the temperature of the water bath

Light Colour

 One group tried changing the colour of light reaching the plant, by placing coloured filters in front of the lamp:

Placing coloured filters between lamp and plant

Counting Bubbles

The rate of bubbles produced was our measure of the rate of photosynthesis.  It was quite tricky to determine because we had to position the plant so that the bubbles were produced at a countable rate, not a stream of tiny bubbles.  Usually one person took charge of timing and another counted.

Using a timer to determine the rate of bubbles produced.

There was lots of effort to take accurate readings and record observations.

January: Rate of Respiration in Living Organisms

I think of this practical activity as "Plants v Snails", but the Nuffield Foundation calls it "Investigate the rate of respiration in living organisms ".

Angie is holding three test tubes containing water and hydrogen carbonate indicator, which changes colour according to the amount of carbon dioxide dissolved in water.  The lighter ones have had exhaled air bubbled through them, so their carbon dioxide concentration is higher as CO2 is partly soluble.  The darker one had only room air bubbled through it.  Hydrogen carbonate indicator is probably the most important indicator you need to know about for biology at this level, so it's a bonus that it gives pretty results.

We used an oxygenating aquarium plant, Cabomba, and some aquarium snails. We could only find tiny snails as the aquarium fish had been eating the indoors ones. The pond was searched but it was so silted up that no live snails were found, so we had to make do with the mini snails!

Two test tubes contained snails only, two had plants only, two both, and two controls had neither. We added some hydrogen carbonate indicator. This did not harm the snails.

One of each type of test tube was put in a dark box, and one in the light. Each set was checked at intervals and the colours of the liquids compared to assess the carbon dioxide concentration.

The tubes in the light containing plants started to change colour around the base of the plants, showing decreased carbon dioxide concentration.

This effect intensified as the plants were exposed to light for longer.

When we took the yellow rack of tubes out of the dark box after a few hours, there was no noticeable change in the colour of the liquid.  No pink was visible, only yellow.  This suggested that the plants had not been photosynthesising.
After a few days, the experiment was ended and the following results were seen:

The tubes have been arranged in order of colour, from the palest yellow on the left to the darkest pink on the right.  This corresponds to carbon dioxide concentration - from highest to lowest.  The plants which had been kept in the dark had respired - given off carbon dioxide - and had not photosynthesised.  The plants which had been in the light had used up carbon dioxide in photosynthesis and the water had turned a rich pink as a result of the hydrogencarbonate indicator.

Our snails did not appear to make much difference.  We think this is because they were too tiny to have a noticeable effect; we coudn't find any bigger ones. The tubes containing snails only may, perhaps, have been marginally paler yellow than the control tubes, but it's hard to say.

In this battle of Plants v Snails, plants won!

Links: 

Nuffield Practical Biology - how do plants and animals change the environment around them?

http://www.nuffieldfoundation.org/practical-biology/how-do-plants-and-animals-change-environment-around-them

January: Testing leaves for starch, and conditions needed for photosynthesis

If we want to tell whether a plant is photosynthesising, we might check whether it is giving off oxygen (as with the pondweed in a tube of water), or we can check whether starch is being produced in its leaves.

Plants produce glucose as a product of photosynthesis.  Just like animals, plants need glucose for respiration and growth.  Glucose is soluble in water and is a small molecule, so it moves easily across cell walls by diffusion.  But for transport and storage, plants need to store glucose in the form of long chains of molecules, known as starch.  Starch is made in the leaves from any surplus glucose not immediately needed.

If a plant is kept in the dark so that it cannot produce new glucose by photosynthesis, it will break down starch stored in the leaves, and in the roots, to release the glucose needed.

Destarching a plant

When we are investigating the conditions needed for photosynthesis, we want to make sure that the plant doesn't have any starch stored in its leaves before the experiment begins, otherwise we won't know whether it has added to its starch stores by photosynthesis.  This is why we 'destarch' the plant first, by placing it in darkness for a period so that it is forced to use up starch stored in its leaves.  For an ordinary geranium houseplant this takes 48 hours.

Testing for Starch

We use the standard food test for starch; we add iodine solution, and if starch is present, a blue-black colour is seen.  A helpful student demonstrated this by dropping iodine solution on some known starchy substances such as cornflour and wheat flour.  He also wrote his name in saliva on some paper soaked in starch solution, and added iodine, to see his letters magically appear white where his spit had digested the starch.

Testing leaves for starch

1. Put the leaf in boiling water for one minute.  This is to kill the leaf, so that it won't be affected by being in the light while we are working on it.
2. Place the leaf in a beaker of alcohol and put this in a hot water bath.  Alcohols boil at relatively low temperatures, so we could see beaker bubbling at 70 - 80C.  The alcohol acts as a solvent and removes the chlorophyll from the leaf, so that it is pale.  The reason for removing the chlorophyll is that its dark colour would make it impossible to see the results of the starch test.
3. Rinse the leaf , pat it dry, and add iodine solution to test for starch.

A freshly picked leaf, and one that has been heated in alcohol.

 This photo shows the alcohol boiling at fairly low temperatures.  It was bubbling slightly at this point, and had just finished bubbling vigorously.

 Our helpful demonstrator is holding a hand boiler next to the beaker.  This is a physics toy - a glass vessel, sealed with low pressure inside, and a volatile liquid which boils at very low temperatures under those conditions.  The heat from your hand makes the liquid boil. We enjoyed watching things bubble away for a while.

 The results of our test were a reflection of the time of year.  The plant which had been kept in the dark certainly didn't have any starch in its leaves.  However, neither did the geranium which had been kept in the light!  We thought that the temperature and light levels were probably just too low for pelargoniums to cooperate.  However, on the tile above you can also see a rectangular strip of aquarium plant, and some of the Cabomba fronds which had been used in another photosynthesis activity, showing traces of blue-black where starch is present.

Links

BBC Bitesize: storage and use of glucose in plants

 Nuffield Practical Science: Testing Leaves for Starch

Nuffield Practical Science: Identifying the conditions needed for photosynthesis (we didn't have time to do this, but it would be a good activity to do on your own).

December: Carbohydrates, Diffusion and Digestion

In this session, we continued looking at sugars and starches, and we saw how starches can be broken back down into simple sugars.  We used iodine to test for starch, and Benedict's Reagent to test for simple sugars. 

What are sugars and carbohydrates?

Carbohydrates are sugars, starches and cellulose, and as the name suggests, they contain only carbon, hydrogen and oxygen.

From the Royal Society of Chemistry's 'Chemistry for Biologists':

"Carbohydrates (also called saccharides) are molecular compounds made from just three elements: carbon, hydrogen and oxygen. Monosaccharides (e.g. glucose) and disaccharides (e.g. sucrose) are relatively small molecules. They are often called sugars. Other carbohydrate molecules are very large (polysaccharides such as starch and cellulose)."

Carbohydrates: rice, honey, sucrose, and glucose

We considered some different types of sugar and looked at molecular models.  Table sugar is sucrose, while the sugar made by photosynthesis is glucose.  Dextrose tablets sold in pharmacies are also glucose (dextrose is just another name for glucose from a natural source). Fructose or 'fruit sugar' is very similar to glucose in structure. 

Monosaccharides are simple sugars, which means they can't be broken down into simpler compounds.  They are single links in a chain.  Glucose, fructose and galactose are common simple sugars.

Disaccharides contain two simple sugars joined together, so they're a chain made of two links. Sucrose is a disaccharide -it contains made from one glucose molecule joined to one fructose molecule. Maltose is another disaccharide and one place this occurs is in the mouth, after we have chewed food and starches have started to break down.

Testing for Sugars - Benedict's Reagent

We introduced Benedict's Reagent (or Benedict's solution), which is used to test for reducing  sugars.  'Reducing sugars'  include all monosaccharides - eg glucose and fructose, and also some 'reducing' disaccharides, including maltose. It does not detect sucrose; sucrose is known as a 'non-reducing sugar'.  'Reducing sugar' just means that 

Angie made Benedict's reagent from copper sulphate, sodium citrate and sodium carbonate.  It is a transparent blue solution which, if heated with reducing sugars, produces a brick - red precipitate.  What this means in practice is that the solution will turn cloudy and anything from yellow to orange to brick - red. 

Honey, glucose and sugar solutions with Benedict's Reagent

We placed samples of glucose (dextrose tablets), honey and table sugar in test tubes with some Benedict's Reagent, and put them in a hot water bath for 5 minutes.  The dextrose tablets and honey produced an orange-yellow precipitate, while the sucrose solution remained transparent blue.

Below is a range of results from the Benedict's test for reducing sugars.  You can see one negative result, which is transparent blue, and colours from yellow to reddish-orange in the positive results.

Range of results from Benedict's test

Positive result appearing!

Aside: the colour change with Benedict's solution is caused by copper oxide precipitating out of the solution, so the blue copper (II) ions, which are soluble, change to red-brown copper (I) ions, which are insoluble.  Because they're insoluble, they form a precipitate.  At the end of the day, some of the weakly positive results looked distinctly coppery when the camera flash reflected off them.  For more on the chemistry of the Benedict's test, see Brilliant Biology Student.

Using Benedict's solution and heating is the classic test for the presence of glucose and other reducing sugars, but a quicker alternative is to use ready-made glucose test sticks.  These are sold so you can test blood or urine for the presence of glucose.  We added honey solution, glucose solution and sucrose solution to some of these, and saw that the colour change was similar to that seen with Benedict's solution.

Saliva breaks down starch!

Alison's initials in spit

Last month we modelled starches built from long chains of sugars.  This time, we investigated how these chains can be dismantled.

We each took a piece of filter paper which had been soaked in rice water - a starch solution.  The starchy filter paper was wrapped around a glass slide. We put saliva on a matchstick and wrote our initials on the filter paper in saliva.  Next, we added a few drops of iodine solution to the filter paper.  The areas containing starch immediately turned blue-black - this is the standard test for the presence of starch, Our initials appeared in white.  Angie had been very much looking forward to showing everyone this activity so was pleased that it was such a hit!  We had some creative patterns drawn in spit. We could have just stopped working at this point and left everybody happily experimenting with spit, starch and iodine!

So what happened here? Saliva contains the enzyme amylase, and this breaks starch down into maltose, which is two units of glucose joined together - so it's breaking down a long chain of glucose molecules into short couples. The starch in our initials was quickly broken down.  We tested that our saliva was working by spitting into a test tube and adding starch solution, then iodine. Initially the solution turned blue-black, but then it began to clear around the saliva. We found that if our test tubes were kept warm, the blue-black colour cleared faster.  This gave us evidence that our amylase could work to digest starch on a larger scale than the filter paper initials. Lachlan's saliva appeared to be very effective!

Visking Tubing as a Model Gut

Next, we tried to model one of the processes which happen during digestion. We made a model gut from Visking tubing, and added some cooked rice (with the cooking water) . The tubing was placed inside a large test tube, which was filled with water to the same level as the rice and water inside the tubing.  This was quite tricky to set up. 

We started by testing the water inside and outside the tubing for starch using iodine solution.  The water inside the tubing contained starch, but the water outside did not. Then we took samples of the water inside and outside and tested for reducing sugars using Benedict's solution. None contained reducing sugars at this stage.  

Testing for starch inside and outside the visking tubing.

Our scientists generously donated some amylase and added this to the visking tubing in the model gut. 

We kept our model guts warm by holding them in our hands or placing them in a warm water bath. At intervals (about 10 minutes) we tested again for glucose inside and outside the tubing.  We took care to keep one pipette for sampling from inside the tube, and one for outside the tube, to avoid contaminating our samples. 

This is what we hoped would be happening, at the molecular level:

Everyone had a positive result using Benedict's solution on the sample taken from inside the visking tubing, but it took a while to find sugars present outside the tubing. Angie only found out the real answer to to this later! See 'Digestion Discussion' below for the full explanation. We considered why this might have been. Why hadn't much glucose diffused through the partially-permeable Visking tubing?  Temperature was likely to be a factor, and also the liquid level had fallen so low in some of the tubes, after repeated sampling for tests, that there may not have been sufficient fluid left in there to allow diffusion to occur. Another possibility was that our rice and starch solution was so concentrated that the starch had formed a coating on the tubing and was blocking diffusion. If we wanted to investigate further, we would need to set up an experiment where we varied just one of these things at a time. The main reason which Angie only worked out later, though, is that salivary enzymes break starch down into maltose initially, and while maltose is a reducing sugar, it is too large to easily diffuse through the Visking tubing. It's great when you solve a mystery!

Starch present inside, but not outside, the tubing, at the start and end of the test.

Model guts in warm water bath, to help our amylase along.

At the end of the day, Angie re-tested the liquid outside the visking tubing and found reducing sugar present outside the membrane in all the models . It all makes sense now!

Hydrolysis of starches

Starch is broken down into sugars by a process called hydrolysis.

From BBC Bitesize Chemistry of Carbohydrates:

Breaking starch down
In animals, during digestion, starch molecules are broken down in the body into small glucose molecules, which can pass through the gut wall and into the bloodstream as an energy supply for the body’s cells. The enzyme amylase is the biological catalyst for this reaction.
Amylase is found in the mouth and gut of animals. The stomach also contains acid, which can also break down starch. The breakdown of starch can be carried out in the laboratory using acid or amylase. Enzymes such as amylase act as biological catalysts in the breakdown of complex food molecules into smaller ones in the digestive system.

Hydrolysis of starch

Starch molecules break down by reacting with water molecules. If any molecule reacts with water molecules to break apart, then this is called an hydrolysis reaction. 

Using acid to break the bonds between sugars

Another way of breaking down starches is by heating them with an acid.  This breaks the bonds between the long chains of sugars.  This process can also break the bonds between simple sugars in sucrose, producing glucose and fructose. 

Angie demonstrated that sucrose solution does not produce a colour change when heated with Benedict's solution, so does not contain free glucose.   However, when sucrose solution is heated with hydrochloric acid, then it tests positive for glucose. The sucrose molecule, which was one glucose molecule joined to a fructose molecule, had been broken down into the simple sugars.

Digestion Discussion

Our model gut represents some aspects of human digestion, but digestion occurs in several stages. We chew food, then saliva acts on it, then stomach acid and enzymes act on it, and finally the gut aborbs nutrients.   Digestion is a big topic and one which you'd need to study separately, but when you look at it, think about our activity and consider how it was similar.

Why did it take so long to find reducing sugars outside the Visking tubing?

We used saliva to help break down starches into components. One component of saliva is the enzyme amylase, which breaks starch down into maltose.  Benedict's test detects the presence of maltose - it is a reducing sugar.  However, maltose is a disaccharide; it is made from two glucose molecules joined together.  This means its molecule size is larger than a monosaccharide, so little, if any, will diffuse through Visking tubing.  Over time, and in the presence of water, and especially if there is an acid environment, the maltose will break down ('hydrolyse') into two glucose molecules, which can pass through the Viking tubing - and some maltose molecules will also diffuse through on their own.  They can 'get lucky', and fit through holes in the selectively permeable membrane if they hit it at the right angle! Think about our molecular models from last time and how a short chain might fit through the net sometimes.

Here's a nice summary of what happens in human digestion: 

Carbohydrase enzymes are secreted by the mouth, pancreas and small intestine.  The carbohydrase enzyme,amylase is secreted by the mouth and found in saliva. It starts to work as soon as we begin to chew our food.  Amylase digests long, complex starch (polysaccharide) molecules, into smaller, simpler maltose (disaccharide) molecules. As maltose is a disaccharide it still needs further digestion before it can be absorbed.  The enzyme maltase breaks it down into glucose.  

In our bodies, salivary enzymes start off the digestive process, along with chewing, but further digestion in the stomach breaks maltose down into glucose, which is the form that our bodies can absorb.  Digestion occurs at more than one site - so our model is very simple compared to what happens in the body.  Glucose does diffuse through the mucosa (mucous membranes inside the gut) in our bodies as it does in the Visking tubing, but as well as this 'passive transport', our bodies also use 'active transport' to take up all of the glucose.  Passive transport would just equalise the concentration either side of the membrane, but our bodies want to get all the available glucose from the food, so we also use something called Active transport, which you can read about in this BBC Bitesize section on movement across cell membranes .  

You can read a critique of the Visking tubing gut model  if you'd like to consider further how this model worked, and how it compares to what happens in our bodies.

Links

Carbohydrates and testing for them - BBC Bitesize

BBC Bitesize: Enzymes and Digestion

Investigating the effect of amylase on a starchy foodstuff | Nuffield Foundation 

Evaluating Visking tubing as a model gut - Nuffield Practical Science 

Starch and glucose - diffusion through semi-permeable membrane

https://www.youtube.com/watch?v=lgWE5m7xUJs

Chemistry for Biologists : Carbohydrates