How to calibrate an eyepiece graticule:

1. Use an eye piece lens that has been fitted with a graticule
2. Place the stage micrometer onto the microscope stage and focus using the low
power objective lens so that the graticule scale becomes superimposed over the
micrometer scale
3. Move the stage micrometer until the start or zero line of each scale is
coincident
4. Look along the scale until another coincident point is found. Ideally, the point
should be as furthest as well
5. The relationship between the two scales can now be calculated
B. Points to focus on while drawing plan diagrams (yes, you could be asked one of
those in P5 as well but not directly from a slide as such)
1. NO SHADING. I think we all know this point well enough
2. NO CELLS in LOW POWER plan diagrams. CELLS NEED TO BE SHOWN in
HIGH POWER plan diagrams
Also, when asked to give magnification, round it off to a whole number. X1000.755
should best be rounded off as x1000
3. Only outlines
4. ANNOTATIONS (Refers to describing the slide for example the xylem is stained
red)
5. Always keep in mind the relative thickness of the various tissue layers being
drawn.

For food tests:

There is only one important point that I would like to make:
If the sample is a piece of food, then grind it WITH SOME WATER in a PESTLE
and MORTAR to break up cells and release the cell contents.
Also to use potato disc in enzyme-substrate reaction: It is best to first
HOMOGENIZE the potato by adding a phosphate buffer of a given pH and then
blending it for 45 second in a high speed blender. Then, filter through cheese
cloth to remove large chunks. Using a paste rather than a thick piece would give
better results.

10 vol H202:
It will produce 10cm3 of oxygen from every cm3 that decomposes. Likewise,
2cm3 10vol H2O2 will give 20cm3 of H2O2
Mass-volume %= Mass of solid solute in g per 100 ml of the resulting solution.
Often used for solutions made from a solid solute dissolved in a liquid

Volume-volume %= Useful when a liquid-liquid solution is being prepared (and for

gases as well). Volume of solute in ml per 100 ml
I don't know why ml is the preferred choice of volume here
Hydrogen peroxide less than 18 vol are low hazard. Solutions at concentrations of
18-28 vol are irritants and any higher concentrations are corrosive.
At all times avoid contact of enzymes with EYES or SKIN. If you happen to come
in contact, wash off immediately with plenty of water.

How to use a colorimeter:

Determine the wavelength of light that the solution absorbs most strongly. This is
simply the complimentary colour of the visible colour of the solution.
Use a colour filter that filters out all but the complimentary colour and set the
wavelength of the colorimeter to the wavelength of the complimentary colour
frequency.
CALIBRATE the colorimeter. This involves referencing. A sample placed in a
CUVETTE is used so that the same glass container with the same absorption is
used all the time to reduce the effect of the container. For calibration, use distilled
water and set the absorbance for that wavelength to be 0%. Alternatively, you
could set the transmittance to 100%
Then place solutions of different known concentrations in the colorimeter one by
one and record the corresponding absorbance/transmission.
Then plot the graph of absorbance/transmittance on the Y axis against
concentration of the solution on the X-axis.
Now, you have a calibration curve
To find the concentration of the unknown solution, place solution in the
colorimeter and read the corresponding absorbance/ transmittance. Now, all you
have got to do is find the corresponding value on the X-axis which gives the given
value of transmittance/absorption on the Y-axis. The value on the X-axis is the
measure of the concentration of the solution!

Practical details of how to prepare a slide of pollen grains and examine

them using a microscope:
1. Brush the pollen grain using a brush and transfer it from the stamen to the
slide.

2. Use a suitable MOUNTANT such as water or glycerol. Iodine solution or

methylene blue can also be used to stain the slide.
3. A COVERGLASS should be placed in position, carefully, in order to avoid air
bubbles being trapped in the preparation.
4. The slide should be placed on the STAGE of the microscope and use the focus
knobs to bring the pollen grains into focus using the fine or the coarse knobs.

Investigation into where chloroplasts are found in a leaf of a flowering

plant:
(I) preparing a microscopic slide of a sample of the leaf
- Let us use the leaf of a maize plant.
- Cut a thin SECTION of the leaf using a RAZOR/SCALPEL
- The mark scheme mentions something about "point of technique such as guard
but I do not understand what that means
-Use forceps to transfer the specimen to the slide or a mounting needle could be
used as well
- Add a mountant such as water on the slide. However, stains such as iodine and
methylene blue can be used as well as determined by:
In a wet mount slide, the specimen is simple held in a drop of water, covered with
a cover slip - it is often used for living material (freshwater protozoa etc).
Some structures are difficult to see using a wet mount and stain is added to the
specimen to show up structures like the nucleus. Stains enhance natural contrast.
- Put cover glass on and be careful to avoid bubbles. For this, lower the glass slide
with support at an angle.
-The slide is then made clean and neat by blotting using a blotting paper.
(II) Setting up and using the microscope:
- Place the glass slide on stage
- Put the clips on
- Illuminate using a light bulb or the microscope or a mirror. Diaphragm of the
microscope is used for light control.
-Adjusting the aperture of diaphragm helps adjust the width of the light beam so
that we are only illuminating the part of the specimen that we are looking at. If
we open it to maximum aperture, we have a lot of stray light from the light bulb
and this decreases the contrast.
-First focus using the low power objective lens.
-Then shift to high power. Changing focus from low to high power is referred to as
zooming.
-In high power, do not turn the coarse focus knob (this determines the distance of
the specimen from the objective lens).
-Only move the fine focus knob.

(III) Recording the observations:

- Drawing - Low power and high power plan diagrams
- High power includes cells as well

Counting stomatal density:

Two ways:
1. Using epidermal strips:
Epidermal strips: The epidermis will peel from some leaves quite readily
- First cut the leaf. We can use our fingernails to catch hold of the leaf and peel off
the epidermis, or we can use a sharp RAZOR BLADE (this sucker keeps turning
up in microscopic slide preparation). Mount the peel in a drop of water on a
microscope slide with a cover slip/
CAUTION: EPIDERMAL STRIPS DO NOT REFER TO THE STRIPS OF A LEAF!

2. Nail Varnish impressions:

- Spread a thin layer of nail varnish on the leaf and LEAVE IT TO DRY.
- Remove the layer of nail varnish by attaching a CLEAR STICKY TAPE to it.
-Peel it off from the leaf and stick it to the slide.
For repetition, be consistent about which part of the leaf to use for the leaves of
the same plant.
-View the epidermal impressions using a calibrated microscope fitted with an Eye
Piece Graticule.
-Calculate the area of the field of view using "(pi) r^2" when we have the measure
of the true radius of the field of view.
-Count the number of stomata impressions visible in each area of impression
sampled.
Now all you have to do is divide the number of stomata visible by the area of the
field of view to get the stomatal density (i.e. no. of stomata visible per unit area of
the leaf).

How to determine the RATE of oxygen uptake

Close the screw clip.
Allow to stabilise.
Note position of manometer fluid and start the clock.
Note the position of the syringe at the point of starting.
Read the position of the fluid at fixed times and after each fixed time, equalize the
levels with the help of the syringe.
Read off volume change in syringe.

Divide the volume change by the fixed interval of time to find the rate of oxygen
uptake.
Do this for series of periods and plot of graph of oxygen uptake against time. The
slope at anytime should give the rate of oxygen uptake at that time.

How to make alginate beads of algae:

(I) why should we go through the trouble at all?
1. Algal balls make it easy to standardise the amount of photosynthetic tissue in
any investigation, enabling semi-quantitative experiments to be undertaken.
2. The algae in the beads will stay alive for several months in a stoppered bottle
of distilled water.
Tips on alginate beads:
- The solution you're mixing the enzyme to be immobilized with is Calcium
Chloride.
- If you want even sized easy to use beads, mix the solution DROPWISE with the
solution containing Sodium Alginate.
- The sodium displaces the calcium in CaCl, giving birth to the calcium alginate
beads
Using the beads:
- Place them in a column like a large burette and use a stand to hold it over a
beaker
- Pour the solution to be broken down by the immobilized enzyme slowly, you want
all of it to be broken down so don't rush them through it!
- As the solution with its broken down contents drops down the nozzle of the
burette, collect it in the beaker
- Before using the apparatus again, run water through the beads to rinse out any
leftover solution
Things to write in the exam:
- When writing about constants, it's better you mention using the same number of
beads per trial and pouring the solution in at the same rate per trial.
- Why is conducting enzyme-substrate experiments in this manner more
convenient? You don't have to separate the enzymes from the reaction mixture
after the substrate has been broken down. The immobilized enzymes are not
easily denatured. They can be reused many times.
How to do it
http://www.eurovolvox.org/Protocols/PDF ... UK_eng.pdf

Tit bits about photosynthesis experiments:

1. to measure light intensity, use light meter/ photo diode/LDR/photometer
2. Hydrogen carbonate is an irritant. So, use gloves and eye protection
3. Do not look directly into lamps
4. Do not touch the lamps while hot
150 W halogen lamps are the best. They have a stand and handle to separate from
the body of the lamp which makes them safer to handle. But they do produce
heat, so we do need a heat filter for investigation.
Heat filter: Use a large erlemeyer (stupidly difficult to pronounce) flask with
water in it. It works as a heat sink with the water absorbing the heat.
Measuring transpiration rate using a potometer:
http://en.wikipedia.org/wiki/Potometer
- Must cut the shoots under water at an angle. If air gets into the xylem vessel of
the plant, it can form air locks that will prevent the plant taking up water and so
reduce the measured rate of transpiration
- Potometers should be left for the leaves to dry. The experiment is not going to
give any meaningful results until any excess water on the leaves has evaporated.
- Adding food coloring to the water makes it easier to see the air bubble in the
capillary tube.
-Use of a reservoir is to reset the air bubble to the end of the capillary tube. For
this, the TAP of the water reservoir has to be opened.
- An air bubble is introduced into the capillary tubing by lifting the whole
potometer upwards. Leave the end of the capillary tube out of the water until an
air bubble forms. Then put the end into a beaker of water.
-WAIT FOR THE BUBBLE TO START MOVING AT A CONSTANT RATE AND THEN
TAKE THE READINGS!
CONTROLS:
Temperature: Increase in temperature increases the KE of water molecules. They
move faster in the xylem. So, there is an increase in the rate of transpiration.
Also, provides the latent heat of vaporisation so water will evaporate faster.
Pressure: Reduced air pressure leads to water evaporating faster and hence
increased rate of evaporation lead to increased rate of transpiration.
Limitations of the procedure:
- The potometer does not measure the rate of transpiration accurately due to the
fact that not all of the water taken by the plant is used for transpiration (water
may be used for photosynthesis or by the cells to maintain turgidity)
POTOMETER ADDITIONAL PROTIP:
Cut the plant underwater to avoid air bubbles from entering the xylem vessels!
Air bubbles block out the passage of water.
Safety:

-Some people find the sap from plants irritating to the skin
-Take care when cutting the plant shoot
-Take care when handling the glass potometer. It is easy to break the long glass
tubes and cut or stab ourselves with broken ends. So, be prepared with first aid
and for cuts from broken glass!
http://www.thestudentroom.co.uk/wiki/Revision:Biology_Practicals__how_to_carry_out_some_experiments

- Whenever there is an experiment that involves measuring quantities and

comparing them, mention the use of a statistical test.
- Whenever you are to clarify the authenticity of a hypothesis using provided data,
the lack of use of a statistical test is usually always a staple point.
- Comparative terms include: Bottom of range of X is larger than top of range of Y
or the range overlaps (for a contradictory argument).
- Whenever you are dealing with something relating to the light-dependent stage
of photosynthesis, make sure you use a lamp at a fixed distance from the
apparatus. Use a bulb of same wattage or use the same light-filter for the whole
procedure (This, of course, is if you are NOT investigating the effect of light on
photosynthesis).
- Whenever dealing with something relating to the light-independent stage,
maintaining the concentration of CO2 in the air is KEY! Mention things like dry
ice and what not (This, ALSO, is only if you are not investigating the effect of CO2
on photosynthesis).
- If it is a potometer experiment, mention both of these. Also mention wind-speed
because that determines the rate of transpiration which then determines the
opening/closure of stomata which then determines the volume of CO2 taken,
which would alter the rate of photosynthesis. If repeating the procedure, take
shoots with the same number of leaves. Use a gas syringe to measure the uptake
of water by the plant.
- For reactions that mention serial dilution, explain how to dilute the solution
(what volumes of water and the solution would be used as an example).
Here's a list of how to keep a couple of variables constant:
Temperature:
- THERMOSTATICALLY CONTROLLED water bath
- Air conditioned room
- Incubator
Wind speed:
- Use of fan
- Fan placed at same distance
- Same Air speed of fan used

To determine the extent to which hypothesis is supported by results,

consider:
1. The general trend shown by the results
2. Any anomalous results
3. The sample size
4. Repeats
5. Range of independent variable for which sample is taken
6. Whether or not tests have been carried out to check the SIGNIFICANCE of the
results
BIO-STATS:
This could have been much better explained by someone who has taken STATS II,
but I unfortunately haven't done that. So , you will have to unfortunately bear
with and CORRECT my misunderstandings.
First of all, we need to realize that irrespective of how scary the terms such as ttest, chi-squared test blah.........., seem all they basically do is test a hypothesis.
The whole of biology stats required in our P5 course deals with HYPOTHESIS
TESTING.
For hypothesis testing we need a hypothesis (duh!)
So, we choose a NULL hypothesis.
The hypothesis assumes that there are no significant differences between the
means of two different samples. A null hypothesis could be: The marks of Pranav
and Mukesh are not different (Or the marks or the same) { For Mukesh: You
always score higher than me buddy so this hypothesis is surely gonna be wrong ,
lol}
All we try to do via statistical tools is to prove that either the null hypothesis is
correct or it is wrong.
Along with NULL HYPOTHESIS, we have an ALTERNATIVE HYPOTHESIS. If the
null hypothesis is proven we assume that the alternative hypothesis is right. It is
to be noted that we do not prove that the alternative hypothesis is right, we just
prove that the null hypothesis is wrong!
Now, let us consider how we can consider how we can prove if the null hypothesis
is right or wrong. To do that, we have to check the SIGNIFICANCE OF OUR
RESULTS.
Please realize that I am explaining what I write to myself as much as I am
explaining it to you.
The all important term: SIGNIFICANCE!
Statistical Significance:
In statistics, a result is called statistically significant if it is unlikely to have
occurred by chance. So, the greater the chance that the result did not occur by

chance , the greater the significance of the result.

How do we check significance:
For that we have got:
1. Chi square test
2. T- Test
( for all the horrible terms, these two are basically all we need to know about)
I am not going to explain the meanings of the term SD and Mean, you MUST
know them by the end of the whole academic year.
So, gonna jump to Chi-Squared test:
Chi-squared test: It is used for DISCRETE data. The probability obtained as an
answer to the chi-squared test states what is the probability that the differences
between the expected results and the obtained results WERE ENTIRELY DUE TO
CHANCE. If the probability is greater than 95, the DIFFERENCES are significant
and the null hypothesis is WRONG. So, if Probability of differences being
significant is HIGH, null hypothesis BYE!
The T-TEST:
ALWAYS KEEP IN MIND THAT THE T-TEST ONLY WORKS FOR CONTINUOUS
DATA! (Unlike the chi-squared test).
First we need to understand standard error:
It is the standard deviation of the sampling distribution of the sample mean. This
distribution is always a normal distribution irrespective of the distribution of the
original sample if n>30 where n is the number of data in each sample
I know it is very confusing , it would be better if you checked out KHAN
ACADEMY videos on this !
Whatever, what we need to understand is , the value of standard error tells us
how close we would expect the means of any further data sets to lie to the first
mean. The smaller the standard error , the more confident we can be that the
means of our second, third and so on data sets will produce means close to the
original mean.
Standard error tells us that we can be 95% sure (this percentage springs from the
property of normal distribution) that , should we the population be sampled again,
the new means obtained will be Mean+- (2X Standard error). If two means lie
within the given range, this is an indication that the two means are not
significantly different.
Standard error is used to calculate confidence limits. These indicate how certain
we can be that the true mean of a whole population lies within the range of
the estimated sample mean
T-TEST:
We use the t-test to assess the significance of the difference between the means of
TWO sets of data which are expected to belong to a normal distribution

For t-test :
1. TWO means are compared
2. The data has normal distribution
3. There is no overlap between sets of data
THE PROBABILITIES THAT THE TEST PRODUCES ARE PROBABILITIES THAT
THE NULL HYPOTHESIS IS CORRECT, AND THERE IS NOT SIGNIFICANT
DIFFERENCE BETWEEN THE MEANS OF TWO SAMPLES.
The t-test (as well as the chi-squared test) have what we call critical values. The
critical value is a value greater than which all t-values would show that the
differences between the two samples are significant. We can think of the tdistribution as a normal distribution when n>30. With the desired t-value(the
mean of the normal distribution) to be zero. The further the value of t from the
given value , the more uneasy the null hypothesis becomes to digest. There comes
one value for which, we have had enough. This value is the critical t-value.
If the total number of observations (both samples added together) is below 30,
error due to chance is significant and the table of t makes an adjustment to
critical values to take this into account, why is why we need to calculate the
HORRIBLE DEGREES OF FREEDOM.
DEGREES OF FREEDOM (WTF!): The degrees of freedom of an estimate is the
number of independent pieces of information that go into the estimate. In
general, the degrees of freedom for an estimate is equal to the number of values
minus the number of parameters estimated en route to the estimate in question.
For example, to estimate the population variance one must first estimate the
population mean. Therefore, if the estimate of variance is based on N
observations, there are N-1 degrees of freedom. For two samples, each with N
samples, the total number of degrees of freedom in N-1+N-1= N-2
It should be noted that t-test gives us a measure of VALIDITY not RELIABILITY
(the extent of reliability is determined by the spread of value about the mean
value i.e. SD). Also, the t-test only compares two means and thus we can only
write comparative statements about those two means. We cannot deduce a
conclusion about the whole sample from a result of t-test which compares only 2
results
I know all that did not make sense, but I tried, sigh... Mukesh you could use your
articulate language to put more sense into these seemingly abstract stuffs!
Alright, let me give an entirely exam-oriented perspective at these statistical
tests:
1) It is just math
2) They give you the formula, there is no memorisation required
3) It assumes you have knowledge on elementary statistics (i.e. mean, standard
deviation)
4) It also assumes that you have brought a reasonably functional calculator with

you
5) It is an excuse you can use when you are asked to suggest to what extent a
given hypothesis is supported by the student's results (trust me, it's always the
student that gets the results, they always use the term student in this type of
thing, coincidence or lack of originality in CIE's part?)
6) They will give you this table for both tests with "degrees of freedom" and the
"probability is greater than". These terms are unnaturally fancy for something this
basic and so are the names "chi-squared" and "t-test". What're you trying to do,
scare us teens from our pursuit of a career in science?
7) Degrees of freedom means the amount of freedom the data has when we are
comparing them. This "freedom" is not the same as the "get out of jail" freedom.
Don't think about it too much, it just means, in our sample of data, there is this
amount of randomness due to the large/small number of data which we need to
keep in consideration when calculating the values for the chi-squared and t
probability. (In other words: the more the data, the more we need to consider the
spread of it, hence we need to alter the testing values to suit the different
numbers of data that may be presented to us) Mathematically, it is just n -1 where
n = number of data.
Calculate --> Compare --> Reject (and by reject I mean reject the null
hypothesis). What a null hypothesis basically is, is the exact opposite statement of
what we are testing. If we are testing the difference between two sets of values,
we say they are not different, so the tests let us prove ourselves wrong (scientists
are so crazy that they love being proven wrong! What a hobby! ). So, we calculate
the t and chi-squared values using the formula that is given to us. Compare it with
the respective value with the same degrees of freedom in the provided table (if
your degree of freedom is not in the table, just find the values it lies between and
find the mean of the respective probabilities of the two values, i.e. your degrees of
freedom is 29 but the table only lists values for 28 and 30. Now if the values at 28
and 30 are 2.1 and 2.2, respectively, then the value you're looking for is 2.15! Get
it?) I don't remember how the value relates to the rejection or acceptance of the
hypothesis but you have Pranav (AKA Zeebu) for that.
9) STAY CALM! This is just a small city of the big country we know as Biology P5,
so please, if you don't understand it, stop wasting your time, move on and
concentrate on better and more important things!
10) Good day!

Points required for planning:

1. Varying the independent variable:
-Suggest how to vary the independent variable
-How the value of the independent of variable will be measured
-Which values of the independent variable will be used (MENTION AT LEAST
FIVE VALUES)
2. Measuring the dependent variable

3. Controlling any 2 variables (you MUST mention the method of controlling). This
mainly deals with the accuracy of the experiment
4. Any 2 procedures of using the apparatus
5. How to make the experiment reliable
6. Safety precaution
if no obvious safety precaution is required you MUST mention that "THIS IS A
LOW RISK EXPERIMENT".
This is awesome stuff. Just use your knowledge in Biology to predict how to
control the variables and how to measure them. Likewise, the constants must be
appropriate to the context. The apparatus will always be something that you have
seen or used before so please make sure you know how to use all the lab stuff.
Safety precaution, don't be skittish to mention your fears of cutting yourself,
burning yourself, corroding yourself (with strong acids) or poisoning yourself (the
dangerously volatile ethanol). Yes, mention "THIS IS A LOW RISK EXPERIMENT".
As a matter of fact, you can say: "Although there is a risk of cutting yourself while
taking a section of <insert appropriate biological content here>, this is a
relatively low risk experiment" to get two whole marks. How's that for cheap?
This is not very important but we can never be sure if CIE picks on this:
Be careful about the use of AGAR and AGAROSE:
Use agar in the context of microbiology
Use agarose in the context of gel electrophoresis
For gel electrophoresis:
http://learn.genetics.utah.edu/content/labs/gel/
If you are asked to reason why a given data is anomalous:
1. First mention that it doesn't fit the general trend and give a reason supporting
that
2. Mention what kind of experimental errors could have resulted in the anomalous
result

Respirometer:
- When using it to compare the rate of respiration of two different species, make

sure the same mass of each species is used in the test.

- When testing the rate of respiration of seedlings, make sure they have not
sprouted green shoots yet (as they will photosynthesize and give back the oxygen
it takes and cheat us of our readings). Nevertheless, if you HAVE to use green
sprouts, conduct the experiment in a dark room or in a container deprived of
light).
- Soda lime and Potassium Hydroxide are two of the best carbon dioxide
absorbents but always make sure to separate them from the individuals of the
species using a wire mesh (keeping in mind the carbon dioxide has to make
through from the organisms to the absorbent but the absorbents should not be
able to come in contact with the organisms as they can be corrosive, poisonous,
etc.)
- Closing the clip on the respirometer to disallow the escape of air and opening it
to reset the apparatus, blah, and the basic workings will give you marks to write
home about.
- The use of a manometer or a gas syringe to measure the decrease in the volume
of air inside the container is a staple to the answer.
- Make sure the gas syringe is completely pulled so that a rather large decrease in
volume can be measured for more accurate results.
- You're supposed to time how long it takes for a particular decrease in volume to
be indicated by the gas syringe or you can also measure how much of the volume
decreases in a particular interval of time (for example: 2 to 3 minutes, more if
you're measuring the rate for something really small or sluggish).
- Also, EQUILIBRATION TIME. You should allow time for the species to adapt to
the environment of the respirometer.
- Temperature should be controlled.
- CLICHE: repeat procedure multiple times and take mean
- Rate of respiration = decrease in volume/time taken
- If you are planning to calculate the respiratory coefficient, remove the carbon
dioxide absorbent (if the total volume INCREASES then the RQ is greater than
one but if it DECREASES it is probably less than one).
-To measure RQ , when the manometer fluid moves away from the tube containing
living organisms, more carbon dioxide is given out than oxygen consumed and if
the manometer fluid moves towards the tube containing living organisms, more
oxygen is consumed than CO2 given out, then by using two formulas calculate RQ.
-For a respirometer, I'd recommend using a manometer with a moving dye
indicator. It is more accurate as no air can pass through the dye at all whereas it
may pass through in a gas syringe. It's all about accuracy and reliability and using
the best suitable apparatus.
Speaking of which, cheap marks galore:
- The volume decrease is determined by measuring the distance moved by dye.
- The actual volume is = l x (pi)r^2 where l is the distance moved by the dye and r
is the radius of cross-section of the capillary tube (the gas syringe is already
calibrated to give volume so there is no opportunity for marks here if you are
speaking of using a gas syringe).