BIOLOGY

LABORATORY REPORT

Name: Nurseiit Alibek

Tutor: Dr. John Carey
Group: F
Lab partners name: Ismagulov Galym
Title: Investigating photosynthesis

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Introduction
Almost every plant takes water and CO2 (carbon dioxide) inside producing sugars or some
complex substances. Waste product of the photosynthesis is oxygen. The total amount of energy
of chemical bonds of the products is more than the total energy in the bonds of the raw materials,
namely water and carbon dioxide. Consequently, this reaction appears to be an endergonic and it
requires some external energy (energy can be taken from the sunlight). Chlorophyll (green
substance) allow the plant to catch light energy that comes from the sunlight in order to produce
sugars [1]. According to Kent (2000, 86), the term photosynthesis can be defined as the process
of using sunlight to build up complex substances from simpler ones. Photosynthesis involves
some complex processes and the simple equation for the photosynthesis can be written as
follows:
light and chlorophyll

6CO2 + 12H2O

C6H12O6 + 6O2 + 6H2O

In addition, every chloroplast has two membranes, which are called the stroma. The stroma has
enzymes, circular DNA and ribosomes, which can be used during the photosynthesis. Membrane
sacs that are disk-like are the thylakoids. When several thylakoids are stacked together they form
the granum. Also, these thylakoid membranes have chlorophyll (photosynthetic pigment).
Moreover, photosynthesis is a very complex process and it has some complex reactions. Also,
photosynthesis has two main stages. They are light-independent stage (dark stage) and lightdependent (light stage). Different books use different definitions to these stages. Obviously,
light-dependent stage occurs only in the presence of light, whereas the light-independent stage
does not require any light. The light-independent stage has the Calvin cycle, where the ATP and
NADPH, which come from the light-dependent stage, are used in order to make some products,
such as carbohydrates or lipids.
This practical aims at demonstrating experimentally the link between the light-independent and
light-dependent reactions. Early in 1937 year, the Robert Hill, who was a biochemist, found out
the fact that isolated chloroplasts could produce oxygen when there is an illumination in the
presence of an electron acceptor. When an electron acceptor (lets assume that electron acceptor
is A) is reduced by protons and electrons, which come from water can be defined as Hill reaction
and can be written as follows [2]:
light

A + H2O

AH2 +

1
2 O2

chloroplasts

In this practical DCPIP (or the 2,6-dichlorophenol-indophenol), which is a blue dye, acts as an
artificial electron acceptor. This artificial electron acceptor (DCPIP) has blue colour in its
oxidized form, whereas in the reduced form it is colourless [2], [3]. Furthermore, centrifuge and
spectrophotometer will be used during this practical. We have previously worked with the
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centrifuge, so we know how it works. However, the spectrophotometer is something new for us,
so we should work with this apparatus very careful and also brief instructions that will be given
by one of the lecturers or the TAs about the usage of this spectrophotometer should be taken into
account.

Safety precautions
During this practical you should wear eye protective equipment (googles), gloves and lab.coat.
Avoid skin contact with DCPIP, since it can stain. In addition, all procedures related with the
centrifuge must be done in the presence of TAs or lecturers. In addition, we should keep the
general safety precautions to avoid any emergency situation.

Materials and Methods

Procedure
This experiment was divided into two parts. Part 1 was isolating chloroplasts and part 2 was
using the chloroplasts. In the first part (isolating the chloroplasts), three small green lettuce,
cabbage or spinach were cut into small pieces using scissors. These small pieces were placed in
prearranged cold mortar, which contained about 20 ml of cold isolation medium. Then, they were
grinded rapidly and vigorously. After that, four layers of nylon were placed in the funnel and
were wet with the cold isolation medium. This mixture was filtered through the funnel with the
nylon into the beaker and then the filtrate was poured into pre-cooled centrifuge tubes, which
were placed in the ice-water-salt bath before. After pouring the filtrate into pre-cooled centrifuge
tubes, the edges of the nylon were gathered and were thoroughly wringed into the beaker and
then transferred into the centrifuge tubes. The centrifuge tubes were checked whether they had
the same volume of filtrate. Then, these tubes were centrifuged for some time in order to get a
small pellet of chloroplasts. This procedure was performed in the presence of the teacher
assistant. After the centrifuge, the liquid (supernatant) was poured off very carefully into the
tube, because we must not lose any pellet during this experiment. This pellet was resuspended
with 2 ml of the isolation medium using glass rod. After that, the procedure such as squirting in
and out of a Pasteur pipette six or five times in order to get a uniform suspension. Finally, this
leaf extract was stored in the ice-water-salt bath and was used as soon as possible in the second
part of this practical. All of the solutions and apparatus, which were used in the first part, were
kept cold in order to preserve the activity of enzymes. Also, the extraction was carried out as
quickly as possible. In the second part (using the chloroplasts), the instructions were read before
the second part starts. Five tubes were labelled and were set up according to the table, which is
represented on the next page.

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Table 1. The tubes were set up as follows:

Tube

Leaf extract
(ml)

Supernatant
(ml)

Isolation
medium (ml)

Distilled
water (ml)

DCPIP
(ml)

0.5

0.5

0.5

0.5

0.5

* means that nothing has been added

After adding the DCPIP to the extract, the tube was shaken and the time was recorded. The tubes
1, 2 and 4 were placed on the place approximately 12-15 cm from a bright light (according to the
manual 100 W). However, the tube 3 was placed in the dark place, namely under the table. All of
the observations were clearly recorded into the logbook. At the same time, another procedure
was performed. After a brief instruction about the use of the spectrophotometer, 5 ml of methanol
was mixed with 200 l of the chloroplast suspension. Then, this solution was centrifuged at high
speed (4000 rpm). Firstly, two small tubes (forgot the exact name of these tubes) were prepared.
One of them contained the centrifuged mixture of the chloroplast suspension and the methanol,
whereas another one contained only methanol. Then, the small tube contained the methanol was
firstly placed into the spectrophotometer and only then the centrifuged mixture was placed into
this spectrophotometer in order to measure the absorbance at 650 nm. In addition, the glass sides
of these small tubes were checked because one side is muddy, so that this kind of thing might
affect the result for the absorbance value. The same procedure was performed at 665 nm. Finally,
the chlorophyll concentration was calculated.

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Results:
Table 2. Observation of the test tubes with different components.
Leaf
Tube extract
(ml)

Supernatant
(ml)

Isolation
medium
(ml)

Distilled
water
(ml)

DCPIP
(ml)

Observations

0.5

After approximately 19 minutes

there was a colour change from
dark blue colour to the pale green.

0.5

No change

0.5

No change

0.5

No change

0.5

No change

* means nothing has been added

Measurement of the absorbance at different wavelengths:

At 650 nm = 1.462
At 665 nm = 1.000

Discussion
According to the obtained results, only first tube, which contained 0.5 ml of leaf extract and 5 ml
of DCPIP, changed its colour from dark blue to the pale green after approximately 19 minutes.
However, at the rest of the tubes there were no change, in other words colour of the solution did
not change. The components in the first and third tubes were exactly the same. The main
difference is that the first tube was placed under the light conditions, whereas the third tube was
placed in the dark place. The change in colour in the first tube happens due to the production of
the NADP2H, so after adding the DCPIP, which detects any production of reducing agents, the
colour of the solution became pale green. Also, as was mentioned before there was no change in
the third tube, since the production of the NADP2H depends on the light. So, this tube was
placed in the dark place, consequently no light and no production of NADPH. As a result, there
was no colour change as it was in the first tube. In addition, the fourth tube did not contain any
DCPIP, consequently there was no change in colour and this was an expected result. The

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remaining tubes, namely second and fifth tubes also did not change their colour, since there was
no leaf extract in these tubes.
Moreover, the chlorophyll concentration can be calculated using this formula:
Concentration (mg/ml) = 0.0255A650 + 0.004A665
The values for A650 and A665 are known. They are: A660 = 1.462; A665 = 1.000
So, Concentration = (0.0255 x 1.462) + (0.004 x 1.000) = 0.04128 mg/ml.
Moreover, let us compare again first and third tubes. First tube was under light conditions and it
changed its colour from dark blue to the pale green, whereas third tube was under dark
conditions and it did not change its colour. In both of the tubes there was a consumption of CO2.
So, it can be concluded that CO2 has a little effect on the reducing capacity of the leaf extract.
Also, CO2 was not involved in light-dependent reactions.
There is a suggestion in order to investigate the effect of light intensity on the light-dependent
reactions of photosynthesis. In order to do so, we should change the distance of the lamp or
change the type of the light. Because if we place the lamp too close to the tubes, we might
destroy some cells by overheating and of course it will affect the final results.

Conclusion
To sum up, during this practical the link between light-dependent and light-independent reactions
were investigated. Also, only the first tube changed its colour, whereas the same tube, which was
placed in the dark condition, did not change its colour. The fourt tube did not change its colour,
since no DCPIP was added and obviously there was no artificial electron acceptor in this tube, so
this result was an expected. Another thing is that second and fifth tubes also did not change their
clour, since there was no leaf extract in these tubes. Moreover, the chlorophyll concentration was
calculated using the data from the spectrophotometer. The concentration was 0.04128 mg/ml. In
addition, in order to investigate the effect of the light intensity on the light-dependent reactions
of the photosynthesis, I think we should change the distance of the lamp, measure this distance
and see the results. Our results seem to be valid and reliable, since they correspond and converge
with our general knowledge about the photosynthesis.

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References
1) Kent, Michael. 2000. Advanced Biology: Photosynthesis. UK: Oxford University Press.
2) http://www.easternct.edu/~adams/Resources/Lab4%20Hill%20Rx.pdf
3) http://www.marietta.edu/~spilatrs/biol309/labexercises/Photosynthesis.pdf

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