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Investigating Into the Possible Existence of Distribution of Stomata Within Different Leaf Types

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Investigating Into the Possible Existence of Distribution of Stomata Within Different Leaf Types

The aim of my particular experiment was to investigate into the
possible existence of distribution of stomata within different leaf
types. My investigation also requires me to research into the rate of
transpiration into the different leaf types and if this has an effect
of the distribution of stomata on the leaves surface.

Prediction

I predict that the environment of which I found my particular leaf
type had an effect on the stomata distribution in my particular leaf
type. From the background knowledge I predict that those plants grown
in a dryer environment and thus must adapt to such climate and within
this situation they will have less stomata on there leaves thus giving
less transpiration from the leaves. The leaves that I obtained from a
dryer environment may have up to less than 150 stomata per 2mm of
leave surface where those in a moist living area have more than 500
stomata per 2 mm.
My prediction also includes that most of the stomata will be found on
the lower epidermis of a leaf. I have based this prediction on the
function of stomata; to let gases in and out of the leaf i.e. to allow
exchange of CO2 and O2 between the inside of the leaf and the
surrounding atmosphere and to allow the escape of water vapour from
the leaf.
To reduce water loss the leaf has a waxy cuticle on the upper
epidermis, which is waterproof, so the leaf uses the lower epidermis
for gas exchange. With such a prediction I would need to carry out the
background research on each particular leaf type chosen to research.

Background Knowledge

Dermal tissue is a kind of complex tissue consisting both of flattened
cells covering the upper and lower surfaces of the leaf and of
specialized cells calledguard cells. Guard cells regulate gas exchange
between the environment and the interior of the leaf by controlling
the size of the stomata, openings through which gas exchange takes
place. The upper and lower layers of dermal tissues are respectively
called the upper and lower epidermis. The epidermises serve both to
protect the plant and for water conservation as the outer surface of
the epidermal cells are covered with a waxy waterproof cuticle.

[IMAGE]

Guard cells within any plant cell:

The guard cells are specialized dermal cells that regulate the size of
the openings or stomata in the epidermis of the leaf. Each stoma is
surrounded by two guard cells that either take up or release water to
the surround cells. Picture:

[IMAGE]

When the guard cells release water to the surrounding epidermal cells,
the guard cells become flaccid which causes the two cells to close off
the stoma. This prevents water loss from the leaf. Conversely, when
the guard cells take up water from the surrounding cells, the guard
cells swell (become turgid) which causes then to bow out, opening the
stoma. This allows gas exchange and an increase in water loss from the
leaf, transpiration.

Water is not directly pumped into or out of the guard cells. Instead,
the guard cells actively transport potassium ions and the water
follows by osmosis.

Guard cells can emit water into three different directions; outwards,
into the neighboring subsidiary cell, and into the respiratory cavity
that is a part of the intercellular system lying beneath the guard
cells.

An equilibrium between the water vapour of the atmosphere and the
respiratory cavity results when the stomata are opened. Plants form an
intermediate distributor, since a large difference in water potential
between the moist soil and the normally dry atmosphere is very common.
They close when too much water is lost, or when not enough supply
exists. The osmotic pressure of the stomata is far larger in the guard
cells than in the subsidiary cells. This ratio shifts in favor of the
subsidiary cells when the stomata are closed.

When the guard cells close their stomata, this conserves water,
something which is important when water is in short supply, but this
also means that carbon dioxide cannot be taken up by the leaf for
photosynthesis and excess oxygen produced by photosynthesis removed.

If the plant is to obtain sufficient carbon dioxide for photosynthesis
it is necessary that the stomata are open, however, in being open,
water can be lost through these same stomata by evaporation. It is
this evaporation, known as transpiration, which is the driving force
for pulling water through a plant. In most broad leaved plants, a
greater number of stomata are found on the cooler, lower surface. This
ensures that sufficient carbon dioxide can enter while at the same
time cutting down the amount of water lost by transpiration.

In this scenario water-uptake can also be preceded by an uptake of
potassium ions. These potassium ions are actively pumped (by a
potassium pump) from the subsidiary cells into the vacuoles of the
guard cells. At the same time, anions (chloride, malate) accumulate
within the vacuoles. Protons are given off to the subsidiary cells.
The ion flows are quantitatively enough to explain a rise of the
turgor that is large enough for the guard cell movements.

A fungal toxin can activate the potassium pump. If consequently the
toxin is applied to the stomata, then the loss of water becomes higher
than its supply resulting in withering.

Water movement through a plant

As transpiration takes place, water diffusing into the air spaces from
the spongy mesophyll cells takes its place. This is turn sets up a
concentration gradient across which water moves by osmosis out of the
xylem cells and across the leaf. A similar concentration gradient
occurs between the xylem and palisade layer so that water will also
move by osmosis to the palisade cells in order that it can be used by
these cells in the food manufacturing process of photosynthesis.

In most plants about 98% of the water taken in by the roots is
transpired from the leaves' surfaces. To give some idea of the
magnitude of water movement, it has been calculated that during the
day a 15 meter high Silver Maple (Acer saccharinum) can lose up to 220
liters of water per hour through transpiration.

Carbon Dioxide & Light:

Plants contain specialized structures within the epidermis of their
leaves which allow for the uptake of carbon dioxide (used as the
carbon source for photosynthesis) and the release of water. These
structures are composed of openings, known as stomata's, and guard
cells which regulate the size of the stomata's. Guard cells respond to
various environmental stimuli by shrinking and swelling which, in
turn, regulates the size of the opening.

Within plants most stomata are closed in darkness yet are stimulated
to open in light. This is similar to the method for plants when
carrying out photosynthesis. This method is most common when looking
at stomata openings but is not the only method, some plants are able
to open at night. Although this is because the particular plant is not
focusing on the amount of light available but the amount of carbon
dioxide available. This carbon dioxide gained during the night is
stored by the plant and fed into the clavin cycle during day time. A
low concentration on carbon dioxide causes the stomata to open; a high
concentration leads to their closing. The activity of photosynthesis
taking place in the guard cells where chloroplasts are found is
related to osmotic value and thus also related to the opening of the
stomata.

Light is able to exert its effects mainly by decreasing intercellular
and intracellular concentration of carbon dioxide. Carbon dioxide is
faster consumed than supplied due to mesophyll's photosynthesis. This
causes an intercellular deficit close to the stomata's opening. The
photosynthetic activity within the guard cells themselves leads to a
decrease of the intracellular level of carbon dioxide, and causes
simultaneously that water is drawn from the subsidiary cells. The more
light available the more stomata you would find located on the
epidermis of the plant.

The number of stomata within a given area of a leaf varies depending
on the species of the plant. The average number of stomata/ cm squaredin
a leaf is about 30,000.There is also often a large difference between
the number of stomata on the upper and lower surfaces of a leaf. For
example, the tomato leaf has approximately 13,000/ cm squaredon it's
lower surface but only 1,200/ cm squaredon its upper surface.

Stomata are commonly located on the lower surface of the leaf, and in
many trees and shrubs they are absent altogether from the upper
epidermis. In other plants where the leaves are found held vertically,
stomata is located on both sides of the stomata. One reason that the
stomata is often only located on the lower epidermis is that the guard
cells and substomatal air spaces would tend to scatter incoming light
if they were on the upper epidermis. As well as this the slightly
lower temperature on the surface may reduce transpiration.

When heated plants may denature to avoid this an individual plants may
open its stomata and evaporate water which will lower the leaf
temperature. One may hypothesize that leaves in the sun should have
higher stomata density than do leave in the shade all else being
equal.

If water is not available, for example in drought conditions excessive
conditions may lead to desiccation and an equal amount of severe
denaturisation taking place in the plant.

Thus one might expect plant leaves exposed to drought conditions have
fewer stomata in sunlit environments. This knowledge I have vacated on
the effects of light and temperature would support and help to prove
my prediction the idea that the environment in which my leaf types
were found would have an effect n the amount of stomata found.

Within this investigation I shall look at the stomata numbers within
the following leaves where I have carried out particular leaf types:

Ivy

There are certain types of plants which have the ability to climb up
various plants and structures. Such plant types are known as climbers
they come from a range of plant families they are nearly all
dictyledons. In most habitats where climber plants are located in
particular dense jungles, the climbing ability is necessary as a means
of reaching sunlight and reaching their full potential in making
photosynthesis to survive. This act as a continuous circle of life for
the climber plants where by the energy saved by the plant by climbing
can be used to enable the plant to grow faster, farther and higher
than neighbouring plants which gives me the plant a positive advantage
over others to be in access of the sunlight and gain even more energy.
The particular leaf type I chose for my experiment from this climber
family was the ivy plant type. I recovered this leaf from my local
school court where it was located carrying out this climber trait upon
a wall structure where it was entwined among other of its leaf type
all competing for available light.

From this above evidence I have vacated a prediction that the Ivy leaf
type having the climbing ability would contain an average amount of
stomata on their leaves since they don't live in really dry or very
moist conditions. Their great requirement for sunlight's which
supplies its energy, suggest that they would have fewer stomata on the
top of the leaf that the bottom which would provide less interference
with the photosynthesis.

Holly Leaves

These are adapted to growing in boreal zones and on mountains, where
there may be a lack of water when the ground is frozen in winter. In
addition their pyramidal shape ensures that snow is more likely to
slide off the branches that collect on then, possibly breaking them.
The conifer leaves are constructed to resist drying out, with a thick
waxy cuticle and, usually small in size.

From such evidence I predict that the holly leaves will have a similar
figure of stomata on both top and bottom epidermis layers of the
leaves. Reasons for such a prediction are that these particular leaves
survive through winter time where sunlight is scarce. Therefore a
large surface area is not necessary for photosynthesis, the larger the
surface area the larger the number of stomata. In addition the area of
the holly leaf is quite moist; they can afford to lose large amounts
of water, meaning their number of stomata is large.

Privet from a bush

Privet is a common ornamental shrub. This plant, a mesophyte, is
adapted to a moderate habitat that is neither very wet nor very dry.

A diagram of the stomata found within Privet Bush:

[IMAGE]

Privet Transpiration:

[IMAGE]

Variegated Holly:

Variegated Holly

Method

A convenient means of explaining stomatal distribution is to make a
replica of the leaf surface using the brush in the bottle. After
applying the nail polish, I will allow it to dry, I then was able to
peel off the replica slowly using forceps. I will then mount it onto a
slide and use a cover slip. By examining the replica slowly under a
microscope, the stomata in a given field of view of the microscope
will be determined by the measuring the diameter with a calibrated
slide or transparent ruler. The amount of stomata will then be
calculated by using the formula πr2 (where r is the radius and the π =
3.142). After gaining a mean value of the stomata found, comparison
between the distribution of stomata in the upper and lower in
different species epidermis will be made.

* Make a replica of leaf surface using the brush in the bottle.

* Allow it to dry, peel off the replica slowly using forceps.

* Mount it onto a slide and use a cover slip.

* Examine the replica slowly under a microscope, the stomata in a
given field of view of the microscope which is determined by the
measuring the diameter with a transparent ruler.

Equipment / Apparatus:

Clear Nail Vanish- This was required in order to make and exact
replica of the plants epidermises clear small and could be easily
peeled off and place onto a slide it was the best replication method
available to me.

Slides- These were required as a base to place my replica upon n order
to see the replica under the microscope.

Cover Slips- These were required in order to keep the replica on the
slide in order to easily see the replica on the microscope so that the
replica nail polish didn't curl up or com off the slide.

Fine Forceps- These were required to peel of the nail vanish once dry
from the leaf surface and placing it onto the slide without it curling
up or having my finger prints over the replica and thus being unable
to see the replica.

Microscope- This was required in order for me to visualise a good
picture of the replica to give me a big enough magnification of the
stomata on each leaf surface.

Materials:

Holly Leaf,

Privet Leaf,

Variegated Holly Leaf,

Ivy Leaf

Fair Test

The variables: In order to make this investigation a fair test, the
test will be carried out on different types of leaves to see if this
will affect the number of the stomata depending on the location the
leaf type came from this will therefore be the only variable to be
changed. There fore the key variables involved include the leaf type
the epidermis layer which I will also be changing and the different
environments which my leaf types came from. By having three different
variables I am able to compare and contrast the distribution of
stomata in each leaf to give e more reliable and accurate results.

I will use the same size slides and cover slips, I will also use an
equal amount of nail vanish on each leaf surface by using one large
sweep of the brush onto the surface.
Three different people will count the number of stomata on each
replication, to get an unbiased number and then an average will be
taken. Only the stomata in the field of view will be counted, to
ensure everyone is counting the same surface area and it is equally
fair for each replication. The same magnification will be used when
viewing under the microscope.

Safety

* Safety goggles were worn when looking down the microscope, to
prevent serious accidents in case someone is pushed.

* All hair should be tied back all lose clothing tucked away and
always wearing an apron.

* Being careful and aware of surroundings at all times, expensive
equipment is being used i.e. the microscope.

* Taking care when cutting the leaf types chosen with pliers no to
damage the plants or your hands.

* Using caution when dealing with the slides and cover slips as
although they are glass still very fragile and if broken can be
very dangerous as sharp.

* Should be aware of the chemicals being used as nail vanish is a
solvent, so you should be careful not to inhale too many vapours
when carrying out experiment.

* When using the particular leaf types one should be careful as some
have sharp edges which are part of their protection in their
initial environment i.e. the holly leaf.

Detailed Procedure for Obtaining Stomata Impressions:

I firstly began my investigation by obtaining the particular leaves
upon which I wished to census stomata. I then decided upon the side I
wished to censer the stomata, typically the leaf underside although my
investigation involved comparing the two and thus I would eventually
censure both sides of plant. I chose to begin censuring the upper
epidermis here I painted a rather thick swath of clear nail polish
upon the surface of the plant. Once the nail polish had dried taking
several minutes, I GENTLY, peeled my nail polish swath from the leaf
using the forceps making sure all nail polish was completely peeled
off the leaf surface. Here I was aware I was carrying out my method of
replication correctly when I saw a cloudy impression of the leaf
surface attached to the nail polish swath. Hereafter my complete swath
could be referred to as my "leaf impression".

I next carefully placed my leaf impression onto the centre of a VERY
CLEAN slide using the same forceps required for peeling off my
impression. I found it particularly effective to use two forceps if
having difficulty placing impression on slide flat and well spread
out. I then used a pen and wrote an ID code signifying the treatment
group name (e.g. the leaf type and which epidermis side impression
taken from) onto a tissue and placed it above the slide. Once the
replication was placed flatly on the slide I then placed carefully a
cover slip over the top to hold the replication in place.

Once cover slip was in place I was required to place my leaf
impression in the centre of the stage of the microscope followed by
placing the metal holders over the slide. Next the important and for
some the more difficult process of this method to focus my leaf
impression under the microscope at least 400x power and observe the
stomata. Next I search around on your impression to find an area that
subjectively appears to have a high density of stomata. That is, move
the slide around until the field of view is away from the edge of the
impression and so that there are no dirt blobs, no thumbprints, no
damaged areas, and no big leaf vein impressions in view.

I subsequently counted all the stomata I see and record the number
neatly on a clearly labeled data sheet. The design of my data sheet on
which I recorded my stomata counts was clearly designed to indicate
which data corresponds to which leaf and epidermis side of plant.
Example of tables used:

Try 1

Try 2

Try 3

Try 4

Try 5

Try 6

Ivy Leaf

Holly Leaf

Privet Leaf

Variegated Holly

Average

No. Found in area of View

Ivy Leaf

Holly Leaf

Privet Leaf

Variegated Holly

I repeat the method six times per epidermis, of which I had four
different leaf types in all I had 96 slides of which I viewed and
counted the stomata counts. From all six stomata counts I recorded I
took an average which was calculated by adding up the results of each
six readings and dividing this sum by six.

You should use a stage micrometer so that you may convert your data
from units of "stomata number per field of view at 400x" to units of
"stomata per "mm²." There are subtle differences among microscopes in
the exact size of the field. You must convert your data to units of
"stomata/ mm²." I carried this out by recalling that the area of a
circle = pi * radius^2. To work out your measurement of the area of
the "field of view" at 400x should be about 0.12 mm^2.

Results!

Epistomatic: Number of Stomata confined to the Upper Epidermis

Try 1

Try 2

Try 3

Try 4

Try 5

Try 6

Ivy Leaf

None

None

None

None

None

None

Holly Leaf

None

None

None

None

None

None

Privet Leaf

None

None

None

None

None

None

Variegated Holly

None

None

None

None

None

None

Average

No. Found in area of View

Ivy Leaf

0

0

Holly Leaf

0

0

Privet Leaf

0

0

Variegated Holly

0

0

Hypostomatic: Number of Stomata confined to the Lower Epidermis

Try 1

Try 2

Try 3

Try 4

Try 5

Try 6

Ivy Leaf

31

27

29

26

22

28

Holly Leaf

42

26

37

27

32

46

Privet Leaf

68

56

53

22

20

26

Variegated Holly

22

25

24

50

74

76

Average

No. Found in area of View

Ivy Leaf

27.16

25.197

Holly Leaf

35

33.037

Privet Leaf

23.16

21.197

Variegated Holly

62.83

60.867

To work out the Magnification for the area of view:

The magnification chosen to be used from the microscope was x40 which
was equal to 5mm diameter. From this information I could work out the
area = πr2 the radius/r is equal to half the diameter = 3.14 x 2.52 =
19.62m2 as we are looking at a x 400 magnification I then divide the
found area by 10 (19.62/10) = 1.962mm2

To work out the number of stomata found in area of view you divide the
average number of stomata sound by the area found i.e. 23.6/1.962 =
12.02mm2

Conclusions& Discussions:

[IMAGE]

As you can see from above graph there were actually no stomata located
on any of the leaf types which is a big difference when compared to
the number of stomata located on the lower epidermises of each leaf
type I investigated.

[IMAGE]

On the above graph you can see that I found a substantial amount of
stomata located on the lower epidermis or the hypostomatic side most
stomata located on the privet leaf at 62.83 rounded to 63 stomata
found, the least number of stomata found was on the variegated holly.

As stated in my conclusion I discovered that out of all four leaf
types the Privet leaf had the most number of stomata on its lower
epidermis. There are several reasons for this these include the
environment in which I took the privet from and the personal
characteristics this particular leaf type has. As stated in my
background knowledge the privet leaf is well adapted to a moderate
habitat that is neither very wet nor very dry. Thus the stomata within
the Privet would be well adapted to opening and closing in the
particular areas where it's wet or dry as the environment in which I
picked the privet was neither dry nor wet in between.

The privet leaf it adapted for the process of photosynthesis with
broad leaved plants, the leaves are large, thin, flat structures.
There leaves are large in fact larger than that of the other leave
type and thus have more stomata, in order to trap lots of light
energy. Their leaves are also thin so that the carbon dioxide can
diffuse into the leaf from the surrounding air. With more carbon
dioxide available in their surroundings compared to that of other leaf
types investigated there is a greater number of stomata simulated to
be within the plant in order to intake the maximum amount of carbon
dioxide available and allow the least amount of transpiration to take
place. The second highest was the holly leaf type at the average
stomata count of 33.037 stomata's per field of view reasons for this
include that they do not have as big a surface area as the privet leaf
as it is not necessary for photosynthesis as plant is adapted to
living in an area of scarce light.

From the graph it can be seen very clearly that most of the stomata
will be found on the lower epidermis, as there is no variation
depending on the type of leaf as the average number of stomata on the
upper epidermis for each leaf type was 0. Although on each of the
lower epidermises there is more than double the amount compared to
that of the upper epidermis the maximum at an average of 60.867 on the
privet leaf. The geranium was the only leaf that did not show a great
variation but a clear conclusion can not be drawn from this as the
number of stomata was only counted once on both the upper and lower
epidermis. The spider plant and begonia had no stomata on the upper
epidermis and although the grape ivy showed an average of 2.5 stomata,
group 1 only counted 1 stomata but group 2 counted 9, a clear
conclusion cannot really be drawn from this because group 2 only
carried out the test once.

My prediction that the greatest number of stomata will be found on the
lower epidermis was proved correct, as was seen by all the leaves
inspected. All the leaves proved without doubt that the greatest
number of stomata are found on the lower epidermis. The ivy, holly,
variegated holly and the privet were all thin leaves without a visible
waxy cuticle so the stomata are located on the lower epidermis to
prevent excessive water loss as they have no waxy cuticle to protect
them. Also they are relatively thin leaves so the exchange of CO2 and
O2 can occur relatively quickly and easily through the stomata of the
lower epidermis.

Evaluation

The sources of error in my investigation could have been; the fact
that different people counted the number of stomata. The error could
have occurred if someone did not know what stomata looked like or they
did not look in the same field of view as the last person. To try and
overcome this error everyone was given a picture of the stomata before
the investigation. Magnification was a variable which was kept
constant, although someone may have adjusted it. Another error in my
investigation included the when peeling the nail varnish, in some
cases it was rather difficult to peel the replication away from the
leaf surface completely. If the replication was not peeled completely
the result would mean a poor slide being produced making it difficult
to see the stomata. There was a chance of mixing up slides, which
would need to be over come if a good investigation was to be carried
out. Other Problems with stomata impressions: some leaves are prone to
damage from the solvent in the nail varnish. The leaves absorb it,
turn brown, and fail to produce any impression. Pupils lose interest
and get frustrated because their leaves 'aren't working'.

Another similar experiment, which could be carried out, is using
cobalt thiocyanate. In the anhydrous state cobalt thiocyanate is blue,
but when hydrated it turns pink. A piece of cobalt thiocyanate paper
is placed on each side of a leaf and sandwiched between two glass
slides clamped together, and then a stop clock started you would
measure the time it takes for the cobalt thiocyanate to go pink as
this indicates that water has escaped out of the leaf. The time varies
in which the colour change takes place depending on the temperature
and humidity. Generally the pink colour develops more rapidly on the
lower epidermis of the leaf than upper surface, the reason already
being discussed in the investigation.

A successful alternative is to use a clear water based varnish. A half
litre tin is cheap, and can be divided up into smaller amounts for
ease of use. Paint the opaque varnish thinly on to the leaf to produce
a clear film. Leave it to dry as usual. This particular water based
varnish takes longer to dry, so if the leaves are coated during one
lesson, the impressions can be peeled off and examined the next. The
varnish is non toxic, so can be used on living plants without removing
the leaves this means that plants do not have to be denuded for this
experiment. In addition to revealing the stomata, the cell walls also
show up for a clearer image.

Other suggestions include producing impressions on acetate film, by
placing a leaf in propanone and then pressing it onto the acetate.
Although this does not work for some plant leaves, especially those
that have an uneven surface and the leaf still has to be removed from
a plant. Another method is to rub a board pen over the surface. The
solvent-based ink permeates the leaf, showing up the stomata. However,
this seems to work only with certain types of pen probably related to
the strong solvent in the pen. Although this raises more severe health
and safety issues.

Further Work:
If this investigation was to be carried out again I would use a
greater variation of leaves, different shapes, sizes, thickness and
leaves from different habitats to see what affect this would have.
Also when peeling off the nail varnish the area would be calculated so
that everyone was counting in the same area also make sure that
everyone repeated the test. Attempts should be made to carry out
similar investigations.

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