Introduction to Investigation Questions

1. Variables of an investigation

• Independent variable: The variable changed by the experimenter (the one

you decide about)
• Dependent variable: The variable measured by the experimenter (the one
you measure)
• Controlled variables: The variables that must be kept constant during the
investigation as they might affect the results. 2. Aspects of an investigation
Accuracy:
• Accuracy can be defined as the difference between actual values and measured
values. The higher the difference the lower the accuracy and vice versa.
• Accuracy is increased by changing the measuring method and using a more
sensitive measuring apparatus.
Validity:
• This means how correct is your experimental procedure.
• Validity is increased by keeping all other factors constant to have a fair test
(controlled variables).
Reliability:
• This means how similar your results are after several replications.
• Error bars/ Range bars show spread of data around the mean. So, they give an
indication on the variability of data.
• The larger the error bars, the wider the data variability and the lower the data
reliability (& vice versa).
• If error bars are overlapping, this reduces data reliability.
• Standard deviation (SD) is similar to error bars but it takes the sample size into
consideration. It is equal sized on both sides of the mean.

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• Reliability is increased by controlling all variables and measuring the dependent
variable accurately.
• Repetition only measures reliability but doesn’t increase it (if repetition by
other scientists gave similar results this enhances reliability)
3. Data tabulation
Independent Variable: comes in the first column in vertically oriented
graphs OR upper row in horizontally oriented graphs.
Dependent Variable: comes in the second column in vertically
oriented graphs OR lower row in horizontally oriented graphs.
Include SI units in the headings of your columns / rows.
You may be asked to calculate the mean = sum of readings divided by their
number
4. Data presentation in Graphs

 Independent variable on X-axis.

 Dependent variable on Y-axis.
 Use more than 50% of both axes.
 Plot all points accurately. Use circled dots or crosses.
 Join points with neat straight lines, don’t extrapolate.  Don’t forget the
units in your axes labels.
Remember:
Your comment on a given graph should include description of the trend, pattern
& data manipulation (subtraction, division or percentage change). Don’t make
theoretical assumptions except if you were asked to explain the changes shown
by the graph.
Don’t comment on minor changes like a small drop or increase between two
points except if it has a clear biological significance
When describing a graph always look for the big picture and don’t talk about
every minor detail. Don’t interpret small changes unless you think there is
definite biological reason for it.

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Some commonly used technical terms:
• Precise results: They are results taken using a sensitive instrument that
measures to smaller increments. (readings with more decimal places)
• Qualitative: a qualitative test tells you what is present. It has the disadvantage
of being subjective.
• Quantitative: a quantitative test tells you how much is present. It has the
advantage of being objective.
• Random errors: unpredictable non consistent errors that occur while taking
measurements. They can affect each individual measurement in a random way
and they affect the pattern of change by producing anomalous results (outliers)
• Systematic errors: quantified consistent errors affecting all measurements. They
don’t affect the pattern of change. They most commonly arise from poor
calibration of the measuring equipment. For example, if a stop watch is faulty
and runs 10% slower than normal, then all timings will be underestimated by
10%.
• Hypothesis: a conclusion based on an observation.

Unit 1 core practical Types

of tests:
1) Quantitative test: tells how much of a certain substance is present.
2) Qualitative test: tells if the substance is present or not with no
indication on quantity.
3) Semi quantitative test: gives an estimate of the present quantity of a
substance.

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Benedict’s test for Reducing Sugars
All monosaccharides and most disaccharides (except sucrose) will reduce blue
CuSO4(II) in Benedict’s reagent producing a precipitate of red Cu2O(I).
-add a few drops of Benedict's solution
-heat the mixture for 2-3 minutes in boiling water bath
-a BRICK RED/ORANGE COLOR is a positive result: glucose is present
-The closer the color is to brick red, the more reducing sugar is present.

Colour changes:
1) Pale blue = no glucose
2) Green = traces of glucose 0.5%
3) Yellow = low level of glucose 1%
4) Orange = moderate level of glucose 1.5%
5) Brick red = high level of glucose >or =2.0%
This is how you would make six serial dilutions of a vitamin C solution,
starting with initial vitamin C concentration of 60 mg cm-3 and diluting each
solution by a scale factor of 2.
1-line up six test tubes in a rack

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 2-Add 10 cm3 of the initial 60 mg cm3 vitamin C solution to the first test tube
and 5 cm3 of distilled water to the other five test tubes.
3-Then, using a pipette, draw 5 cm3 of the solution from the first test tube,
add it to the distilled water in the second test tube and mix the solution
thoroughly. You now have 10 cm3 of solution that’s half as concentrated as
the solution in the first test tube (its 30 mg cm-3).
4-Repeat this process four more times to create solutions of 15 mg cm-3, 7.5
mg cm-3, 3.75 mg cm-3 and 1.875 mg cm-3.

To be able to estimate concentrations of reducing sugars using Benedict’s

reagent
Plan how you will use the stock 4% glucose solution to make the following five
concentrations of glucose solution: 4%, 2%, 1%, 0.5% and 0.25%.....serial dilution
method.
1. Use 4% glucose solution and distilled water to creat 10 cm3 of 5 solutions with
different known glucose concentrations (4%, 2%, 1%, 0.5%, 0.25%) using serial
dilution.
2. Get six test tubes and label using water proof pen.
3. Add in each test tube 2 cm3 Benedict’s solution.
4. Add 1 cm3 of each known glucose solution using a clean syringe in the
corresponding labelled test tube, and put 1 cm3 of unknown sample in the
sixth test tube.
5. Allow gentle shaking to ensure mixing.

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6. Heat all test tubes in a water bath at 80°C for 2 minutes using a stop watch.
7. Use a tong to remove test tubes from water bath and place on test tube track.
8. For Semiquantitative method:
Where you have prepared a range of colour standards ( colour chart) with known
concentration of glucose to compare the results of test carried out (juice of
unknown sugar concentration) with an equal volume of known solution.

Testing for proteins

Add the food sample to water and mix carefully.
Add a known volume of Biuret’s reagent.
If colour changes from blue into purple, proteins are present
(Positive test)

Test for Starch

The brown Iodine solution reacts with starch and changes it to a blue-black color.
This test helps you to find out if a food contains starch.
-add Iodine solution to a solution or directly onto materials such as bread, potato,
crackers...
-a BLUE-BLACK COLOR is a positive result: starch is present
Explanation
-Starch can be separated into two fractions- amylose and amylopectin.
-Amylose in starch is responsible for the formation of a deep blue color in the
presence of iodine.
The iodine molecule slips inside of the amylose coil to give a special color.

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How are these tests semi quantitative?
To have a semi quantitative test a colorimeter is used to measure the
absorbance of colour or transmission of light.
By detecting the colour intensity we can estimate the concentration of
substance X by using a calibration curve which shows the intensity of colour
(on the Y-axis) plotted against the concentration of substance X (on the
Xaxis).
The deeper the colour, the higher the concentration of substance X.

 To be able to estimate concentrations of starch using iodine

solution:
1. Plan how you will use the stock 2% starch solution to make the following five
concentrations of starch solution: 2%, 1%, 0.5%, 0.2% and 0.1%...same as
previous experiment.
2. Use the waterproof pen to label five test tubes and five small beakers with the
different starch concentrations they will contain. Label the sixth test tube
‘unknown’.
3. Use the syringes, the distilled water and the 2% starch solution to create 5 cm3
of each solution in the corresponding labelled beaker.
4. Use a clean syringe to add 0.5 cm3 of iodine to each of the six labelled test
tubes. Add a further 10 cm3 of distilled water to each tube.

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5. Using a clean syringe each time, add 5 cm3 of each starch solution to the
corresponding labelled test tube.
6. Add 5 cm3 of the ‘unknown’ starch solution to the sixth test tube.
Compare the colour produced in this tube with the colour of the diluted test
tubes.
7. Record your results in a suitable table.
8. Then use same semiquantitative method as before

Investigating Vitamin C content of different juices

 Add 2 cm3 of 0.1% DCPIP to a test tube.
 Prepare juices from both fruit species using the same extraction method.
 Use a pipette to add equal sized drops of the tested juice using a pipette to
the test tube containing the blue DCPIP solution.
 Shake well after each drop.
 Records the number of drops needed to change DCPIP from blue to
colourless.
 Repeat the process and calculate the average.
 To compare vitamin C concentration of different juices, use number of
drops as a reference.
 The higher the number of drops needed to decolorize DCPIP, the lower is
vitamin C concentration and vice versa.
 To increase accuracy, use a calibrated pipette to measure the exact volume
of juice needed to decolorize DCPIP.
To know the exact vitamin C concentration of the tested juice you can use a
calibration curve OR repeat the process with a standard vitamin C juice (solution
of known concentration) & record the results then use the following equation:
Conc. B = (Vol. A * conc. A)/ vol. B Vitamin C act as an
Vol. A = volume of Vitamin C solution in ml antioxidant that
reduce plaque and
Conc. A = Concentration of vitamin C solution in mg/ml
atheroma formation
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Vol. B = Volume of fruit juice in ml
Conc. B = concentration of vitamin C in fruit juice in mg/ml

Safety:
-Wear eye protection.
-Avoid skin contact with any reagent.
-Handle the test tubes with tongs to avoid burns

Possible evaluation issues

-Difficulty in controlling temperature.
-Amount of shaking (too much adds oxygen which will slightly restore the DCPIP to blue)
-End point difficult to judge as needs to be just when blue colour disappears especially in highly
coloured juices.
-Some loss of solution when transferring from one beaker to another.
-Accuracy of measuring equipment.

1. What were the independent and dependent variables in this investigation?

The independent variable was the type of fruit juice used. The dependent variable was the volume
of juice added to decolorize the DCPIP.
Important questions

2. Why was each titration completed three times to calculate a mean?

Calculating a mean value makes it easier to spot anomalies. Repeated readings also reduce the
effect of errors.

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3. Suggest one reason why syringes were used in this investigation rather than
burettes:
• Using very small volumes of solution requires a more precise piece of apparatus than a
burette.
• Syringes are easier to control, allowing smaller volumes to be added.
• Ease of use
• Relatively low cost

4. Which of the fruit juices tested contained the highest concentration of vitamin
C?
Your own answer according to your results

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Effect of temperature on cell membranes
 Cut equal sized pieces of fresh beetroot using a cork borer.
 Rinse all discs under fresh water for 5 minutes to remove any pigment
produced from cutting.
 Add six cubes of beetroots to a test tube.
 Add 5 cm3 of distilled water to the test tube.
 Prepare a water bath set at 10 oC using a large beaker, tripod, gauze and
Bunsen burner (wear goggles) and place the test tube in it.
 Leave beetroots for an hour.
 After the hour, shake the tube and hold it to light to observe the color of
the solution.
 Repeat the exact same procedure for other test tubes with temperatures
20, 30, 40, 50 & 60 oC
 Compare colors of solution in different test tubes.
 To improve accuracy, use a clean cuvette & a colorimeter which could
either measure transmission of light or absorbance of color.
 To ensure validity, control all variables such as… Independent variable:
Temperature of water.
Dependent variable: Absorbance of color from the resulting solution.
Controlled variables:

 Volume of distilled water in the test tubes.

 Number of beetroot cubes in the test tubes.
 Time left in water.
 Size of beet root cubes.
 Storage age and storage conditions of beetroots.

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Expected outcome:
As the temperature increases, the intensity of red color increases.
Beetroots have Betalain pigment stored in their vacuoles, High temperatures
causes coagulation of membrane proteins & melting of the phospholipid bilayer
leading to loss of selective permeability and escape of the Betalain pigment from
the vacuole into cytoplasm then outside the cells into water.
N.B. Alcohol also causes loss of membrane permeability as it dissolves the
phospholipid bilayer. To investigate the effect of alcohol on membrane
permeability, the same investigation is repeated but with using a range of alcohol
concentrations and fixing the temperature.

Explain how (a) high temperatures and (b) ethanol damage cell
membranes. Make reference to the fluid mosaic model in your answer. (4
marks) Any four from:
1. Cell membranes (surface and around organelles, e.g. vacuole) consist of
proteins
2. floating in a phospholipid bilayer. (1)
3. High temperatures can denature the proteins by disrupting the bonds that hold
their tertiary structure in place. (1)
4. High temperatures increase the fluidity of the phospholipid bilayer and may
melt the lipids, causing gaps in the bilayer. (1)
5. Ethanol at high enough concentrations may denature some proteins by
altering hydrophobic interactions. (1)
6. Alcohol disrupts the phospholipid bilayer and this is more severe in plant cell
membranes as they lack cholesterol which helps stabilise the membrane. (1)

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The cellulose cell walls of plant cells are permeable whereas the cell
membranes of plant cells are partially permeable.
Explain the meaning of the terms
(a) permeable and
(b) partially permeable. (2 marks)

Permeable: allows any type of / size of molecule to pass through

Partially permeable: only allows some molecules (of certain size or type) to
pass-through

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Effect of Changing Substrate Concentration on the Rate of Reaction

› Use a large syringe to measure 20 cm3 of pureed potato into a conical flask
› Connect this conical flask using a delivery tube to a half-full water bowl with an inverted
measuring cylinder.
› Measure 2 cm3 of Hydrogen peroxide using a syringe and add them to the pureed potato.
› After 30 seconds note the volume of oxygen in the measuring cylinder.
› Divide the volume of oxygen given out by 30 to get the initial rate of reaction in terms of cm3/S

› Repeat the whole process using different concentration of H2O2 such as 30, 40 & 50 cm3 ›

Record the results in a table.

› Plot a graph showing the effect of changing H2O2 concentration on the rate of reaction.
› To ensure validity, control temperature using a thermostatic water bath and control pH by
adding buffer solution.
› To ensure reliability, repeat several times for each concentration of catalase and calculate the
average rate of reaction.

Why use catalase?

1) Abundant in nature as it’s found in most living organisms.

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2) Its an enzyme with very high specific activity.
3) The product is oxygen gas which is easy to measure.
Independent variable: substrate concentration
Dependent variable: Initial rate of reaction at each concentration.
Controlled variables:
› Temperature
› pH
› Volume & concentration of the enzyme.
› Volume of the substrate
To investigate the effect of enzyme concentration on the rate of reaction, repeat
the same investigation but with varying the volume of pureed potatoes used and
fixing H2O2 concentration.
Ideally, you would repeat the procedure for each factor several times. Explain
why it is important to measure the initial rate of the reaction rather than an
average rate over a longer time period. ?
Because the reaction is rapid and the milk (substrate) concentration quickly
declines. The rate slows as the substrate is used up. Therefore, it is only possible
to make valid comparisons at the start of the reaction, when controlled variables
such as substrate concentration are the same for all levels of the independent
variable.
If the surface of the cuvette is scratched, this can result in a greater absorbance
of light.
If the cuvette used for the reaction was scratched (but the reference cuvette was
not), would this give a random or a systematic error? Explain your answer. • A
systematic error, because it would cause absorbance readings to be higher than
the true
Explain the term ‘serial dilution’ (2marks).
A stepwise dilution of a solution (1)

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The dilution factor at each step is constant / the concentration decreases by the
same factor with each step. (1)
Explain the advantages of using serial dilutions. (3 marks) The
dilutions cover a wide range.
Dilutions are easy to carry out as the steps are similar in each case.
Dilutions cover the range evenly

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Investigating Mitosis by squashing Root Tip

Important Safety precautions:

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1. Cut away from yourself to prevent injuring your hands.
2. Be careful while handling the microscopic slide as it is made of fragile
glassware so it might break cutting your hands.
3. Wear eye goggles to protect your eyes from HCL & stain.
4. Wear a lab coat to protect your clothes from the stain.

Different cell cycle phases as viewed under the light microscope

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Zones of the Root
• Protective root cap: constitutes the terminal
1mm of the root tip and formed of dead cells.
• Zone of cell division: the cells are dividing and
increasing in number (Not size).
• Zone of cell elongation: the cells are growing
and increasing in size (Not number).

Place root tip in hydrochloric acid to separate the cells/ macerate tissue/ soften
tissue (making it easier to stain)
Addition of appropriate stain such as toludine (blue) , acetic orcein, methylene
blue to high light chromosomes.
Place the root tip on microscope slide, teasing cells (using needle to spread cells),
Squashing the cells underneath a cover slip to separate cells (for easier view), and
heat the slide to intensify the stain.

Ensure the students view the preparation under the microscope on low power
first to identify the area of dividing cells and to focus. Higher power will be
required to examine the dividing cells for the stages of mitosis
# suggest why a stain was used on the stem and root cells?
-The thin section of stem and root do not absorb much light, so they are difficult to
see using a light microscope. The stain absorbs more light, making it easier to see
the structures. Not all of the cells are stained so difference can be seen

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 Measuring Tensile Strength of Plant Fibers

Independent variable: Type & source of plant fibre.

Dependent variable: maximum weight the plant fibre can hold (tensile strength)
Controlled variables:

• Same age of plants and same storage conditions.

• Fibers should be of the same length at the start and same diameter.
• External conditions as temperature of the room or humidity.

 N.B.: Tensile strength:

It is the maximum stress a material can withstand without breaking.
To measure tensile strength:
• Record the average mass at which the fiber breaks after several
repetitions.
• Covert mass into force
• Divide the force by the cross sectional area of the investigated fiber.

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Investigating Effect of Mineral Deficiency on Plant Growth

Independent variable: Deficient mineral (minerals in the solution)

Dependent variable: physical characteristics of the plant Controlled
variables:

• Age & species of the used seedlings.

• Volume of water in the solution.
• Temperature.
• Light intensity.

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Precaution: Use sterile tubes to avoid bacterial or fungal growth.
Importance of minerals

• Nitrogen: formation of amino acids for protein synthesis.

• Calcium: formation of middle lamella “calcium pectate”.
• Magnesium: formation of chlorophyll pigment.
• Phosphate: making ATP, DNA and RNA How to measure the Dry mass?
Place the plant in an oven at temperature 80 C for 24 hours, this removes all the
water content. Then, measure the mass using a sensitive balance until you get
three successive similar readings.

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Investigating The Antimicrobial Effect of Plant Extracts

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Independent variable: Type of plant extract
Dependent variable: Zone of inhibition around the hole / disc.
Controlled variables:
› Volume of plant extract in each hole
› Concentration of plant extract
› Bacterial lawn on the petri dish

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Explain briefly how cells can be measured using a microscope. A suitable
summary might include the following points.
- Use a stage micrometer and eyepiece graticule (eyepiece micrometer).
Calibrate the eyepiece graticule with the objective lens that will be used.
Do this by measuring the eyepiece scale against the scale of the stage
micrometer.
- Measure the cell using the eyepiece scale and convert into length units (µm).

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Suggest improvements to the method that was used to describe the size of cells
in a tissue.
• Calculate a mean. Measure more cells to estimate a more reliable mean.
• The volume of the cells would be a better descriptor of size than the linear
dimensions.
Under a high-power objective lens, 300 µm on a stage micrometer was Found to
be equivalent to 87 eyepiece units.
(a) What is the size of each eyepiece unit in µm? (1mark)
300 divide 87 = 3.4 µm (1) Allow
3.45 / 3.5 µm.
(b) Using the same objective, the diameter of a red blood cell was measured
as2.5 eyepiece units. What is the actual diameter of the red blood cell? (2 marks)
2.5 × 3.4 = 8.5 (allow in range 8–9) (1) µm (1)

Magnification is how many bigger is the image compared with actual size while
Resolution is the smallest distance that two objects can be apart while still appearing
as two objects.
In other words, resolution allows us to see more fine details.

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 • Human eyes have limited magnification & resolution so to observe biology we
need to increase magnification and resolution using microscopes.
• The resolution of the human eye is 0.1 mm.
• The eukaryotic cells are roughly 1- 20 um in size while prokaryotic cells are 0.1 –
5 um in size.

The Light Microscope (The Optical microscope)

• The first microscope by mankind and the most commonly used.

• Light is sent from a light source then passes through the specimen and then
the image is magnified by glass lenses.

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They are the most common as:

• They are cheap.

• They are Easy to use.

• They can be used to study Living cells so can view

processes occurring in real time.
However,

• Light microscopes produce 2D images.

• They use visible light to create an image, this limits

their resolution to 200nm and magnification to 2000x.

The Electron Microscope

• Instead of using light beams, the electron microscopes use beam of electrons to
study specimens.
• Electrons have a shorter wavelength so they can create images with higher
resolution.

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 • There are two types of electron microscopes: scanning electron microscope
(SEM) and transmission electron microscope (TEM)
• In both types a beam of electrons is focused by a magnet towards the specimen.
• Specimen must be coated with metal and placed in vacuum. This preparation
means electron microscopy can only be used to study dead specimens.

SEM: After the electrons hit the specimen they become scattered on a detector. This
scattering allows to determine how far away are structures in the specimen from each
other and thus it produces a 3D image. However, the scattering of electrons reduces
magnification and resolution.

TEM: Electron beam travels through the specimen to a detector below with no
scattering. This allows for higher resolution and magnification, but produces a 2D
image.

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Size Units
To make proper use of magnification calculations we need to know how to convert
between size units. The SI unit for distance is a meter. The units we are interested in are
the: meter, centimeter, millimeter, micrometer and nanometer.

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1m = 100 cm
1cm = 10 mm
So
1m= 1000 mm
1mm= 1000 um
1um= 1000 nm

Calculating Magnification
1) Write down the equation:
2) Image = Actual X Magnification
3) Covert given into the same units
4) Substitute in the equation

Staining cells for Microscopy

What is staining?
• Staining is a technique where dye is used to highlight cells in the microscope.
• Some structures already modify light as it passes through them so there is no
need to stain them as they are colorful. Example: Algae that contain chlorophyll
pigment.
• The stain absorbs more light, making it easier to see the structures. Not all of
the cells are stained so differences can be seen.
• We only stain when we need to view structures that we wouldn’t normally be
able to see as they are transparent.

How stains work

Stains work by collecting around particular types of
molecules.
For example, acetic orcein is a stain that collects
around DNA producing a red colour.

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Metachromatic stains: They are differential stains that
highlight different components of the cell with
different colours. Iodine stains starch grains blue
black while other cellular components appear yellow.

The all-purpose metachromatic stain is Toluidine

blue O (TBO). For example, TBO stains lignin blue,
pectin pinkish & nucleic acids purple.

Observing the slide occurs through the following steps:

• Examine by naked eye first to be able to position the slide correctly on the
microscope stage
• Select the lower power lens first.
• Place the slide on the stage of the light microscope and move it so the
specimen is directly under the lens
• Switch on the microscope light to shine through the specimen.
• Use focus wheel to bring into focus
• Scroll the stage around to view different areas of the specimen
• Move to the next power lens to view things more specifically and more
detailed.

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 • Use the highest power lens to observe in more details and the shift to the
lower power whenever you need to reposition the specimen.

Plant tissue preparation: (stem, root or leaves) Procedure

:
1. Lay the stem on a chopping board
2. Use a scalpel to cut transvers sections of the
stem. (Make sure that the sharp end of the
scalpel is facing away from you to avoid
cutting yourself & wet the scalpel to reduce
friction so that you don’t damage the tissue)
3. Collect a thin section of plant stem. Add a
few drops of water to the centre of the
white tile
4. Use a mounted needle/forceps to lower the
coverslip onto it gently.
5. Add two drops of toluidine blue O stain and
leave for 2-4 mins.
6. Then add cover slip and gently remove
excess stain with a paper towel.

Observing samples of animal cells:

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N.B. Always wear gloves when handling samples to prevent bacteria on the hand
from infecting the specimen. Also use a sterile forceps to transfer the specimen
to the slide as this prevents pathogens reaching the sample & also prevents
distortion of the sample.
Errors:
-in slide preparation: sample contamination, artefacts, wrong staining, dirty
slides/lend
-errors in calculations or inability to use graticule

Volume of cylinder=r2h

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