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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 t-test, 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 t-distribution 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).