54

Introduction to Investigation Questions

Important Terms
› Accuracy:
• This means how correct your measurements are.
• To ↑ accuracy: change the measuring method and use more sensitive measuring
apparatus (sensitive measurements).
› Validity:
• This means how correct is your experimental procedure.
• To ↑ validity: keep all other factors constant to have a fair test (controlled variables).
› Reliability:
• This means how similar your results are.
• To ↑ reliability: replicate your experiment and get average.
Data Organization
Tabulation
› Independent Variable:
It is the variable controlled by the experimenter, it comes in the first column (left and upper / X-
axis).
› Dependent Variable:
It refers to the measured results which depends on the independent variable, it comes in the second
column (right and down).
› Label all columns and rows.
› Include SI units in the heading of your columns and rows.
Remember:
𝑆𝑆𝑆𝑆𝑆𝑆 𝑜𝑜𝑜𝑜 𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟
› you will be asked to calculate the mean =
𝑇𝑇ℎ𝑟𝑟𝑟𝑟𝑟𝑟 𝑟𝑟𝑆𝑆𝑆𝑆𝑛𝑛𝑟𝑟𝑟𝑟
Graph
› Independent variable on X-axis.
› Dependent variable on Y-axis.
› Use more than 50% of the graph.
› Plot all points accurately. Use circles, crosses or dots.
› Join points with neat straight lines, don’t extrapolate.
› Don’t forget the units in your axes labels.

© Dr. Mohab Megahed

 55

Unit 2 Core Practicals

1) Investigating Mitosis by Root Tip Squash
Root structure
› Protective root cap: Zone of dead cells (1mm)
› Zone of cell division: cells are large in number but small in
size (Mitosis)
› Zone of cell elongation: cells are large in size but small in
number

Practical steps
1) Cut the terminal 1 cm of onion root using a scalpel.
2) Transfer to a watch glass containing 2 cm³ of HCl. This softens the tissue by dissolving the
middle lamellae which is a glue-like material holding cells together.
3) Transfer using forceps to a microscopic slide with a drop of stain (Examples:
Acetocarmine, Acetic orcein, Feulgen stain, Schiff’s reagent, Toliudine blue). This
highlights chromosomes to be visible.
4) Use a scalpel to cut the tissue again into smaller pieces. This increases surface area for
better uptake of stain by diffusion.
5) Cover the tissue sample with a cover slip.
6) Insert the slide into a slide warmer. Heat provides kinetic energy to intensify the stain by
increasing the rate of diffusion.
7) Insert the microscopic slide over filter paper. Using your hands, apply gentle pressure
vertically downwards. This is known as squashing which is needed to flatten the sample
and separate cells to be easily viewed under the light microscope (Avoid lateral movement
of the cover slip to prevent overlapping of cells which damages the sample)
8) Place the slide on the stage of a light microscope. Use the low power lens to scan & detect
the area of mitosis then the high power lens to view mitotic features.

© Dr. Mohab Megahed

 56
Precautions
› Cut away from yourself to avoid cutting your skin.
› Wear safety goggles to protect your eyes from the HCl used in the investigation.
› Wear a lab coat to prevent the stain from affecting your clothes.

Value of the experiment

› To observe the mitotic features.
› To calculate mitotic index =
(Number of cells undergoing mitosis / Total no. of cells viewed) x 100
› To estimate the duration of each phase of mitosis; the number of cells in a certain phase is
proportionate to the duration of the phase. The larger the number, the larger the phase .

Homogenous short interlacing cells aligned V-shaped 2 nuclei are

nucleus threads at the equator chromosomes seen

© Dr. Mohab Megahed

 57
2) Investigating tensile strength of plant fibers
Tensile strength: The maximum stress a plant fiber can withstand without breaking.
Investigation design
1) Select plant fibers of the same initial length
& diameter.
2) Suspend gradually increasing masses to the
plant fiber.
3) Record the mass at which the fiber breaks.
4) Convert the mass into force then convert the
force into tensile strength.
5) To ensure accuracy, repeat with narrower
intervals between the last 2 masses added.
6) To ensure validity, keep other factors
constant such as temperature & humidity.
7) To ensure reliability, repeat for each type of
fiber & calculate the average tensile strength.
How to control variables
› Initial length: Use a measuring tape to ensure all fibers have the same initial length.
› Initial diameter: Use a micrometer or Vernier caliper.
› Temperature: Use a temperature controlled room with AC.
› Humidity: Same closed chamber (with humidifier).

Vernier caliper

© Dr. Mohab Megahed

 58
Safety precaution
› Wear safety goggles to protect your eyes from the fiber after it breaks.
› Use a soft matt (wet fabric) under the suspended mass to protect your feet.
› Tie a rope around your waist while collecting fibers from plants to protect yourself from
falling down.
Advantages of plant fibers (plant based products VS. oil based products)
› Sustainable: As plants can be regrown unlimited number of times so they never run out
like oil based products
› Biodegradable: Broken down by bacterial enzymes in decomposition so they don’t
accumulate in nature.

3) Investigating the effect of mineral ion deficiency

Investigation design
1) Select maze seedlings of the same species, age &
original mass/length.
2) Place a seedling in a beaker containing a solution of
all mineral ions except nitrate ions.
3) Close the beaker with cotton wool. This allows the
entry of air supplying oxygen & reduces entry of
pathogens.
4) Wrap aluminum foil around the beaker to prevent
entry of sunlight. This stimulates(mimics) soil
conditions & prevents overgrowth of algae.
5) Leave for 4 weeks then measure the final mass of the
seedling.
6) Calculate the change in mass.
7) Repeat the whole process using another seedling placed in a solution containing all mineral
ions including nitrates. This is a control for comparison.
8) To ensure validity, keep all other variables constant such as temperature, light intensity,
humidity, pH of solution and concentration of mineral ions.
9) To ensure reliability, repeat 3 times for each type of solution and calculate the average
change in mass.

© Dr. Mohab Megahed

 59
How to control variables
› Temperature: temperature controlled room with AC/thermostatic water bath
› Light intensity: use light bulbs of the same voltage (wattage) at the same distance
› Humidity: same closed chamber/tie a clear plastic bag around the shoot
› Solution pH: use buffer solution to keep constant pH
› Mineral ion concentration: add known mass of each mineral ion to a known volume of
water
Importance of mineral ions
› Nitrate ions Amino acids Proteins Enzymes Photosynthesis Better growth
› Magnesium ions Chlorophyll pigment Traps sunlight Photosynthesis Better
growth
› Calcium ions Calcium pectate in the middle lamella Holds microfibrils of adjacent
cell walls together Higher tensile strength Better growth
› Phosphate ions ATP energy Better growth
DNA} more mitosis Better growth
RNA} more protein synthesis

© Dr. Mohab Megahed

 60
4) Investigating antimicrobial properties of plant extracts
Practical steps
1) Spread the bacterial culture evenly over the surface
of agar. (lawn distribution using pipette or
streaking method using inoculating loop)
2) Make 2 equal sized holes in agar.
3) Use a pipette to add 0.1cm3 of maize extract to one
hole & 0.1cm3 of water to the other hole. (control
for comparison)
4) Cover the petri dish by a vertically fixed lid.
5) Incubate at temperature of 28°C for 24 hours.
6) Measure the surface area of inhibition zone around
the wells/ holes using graph paper tracing.
7) The larger the inhibition zone, the higher the antibacterial properties.
8) Repeat 3 times for each extract & calculate the average area of inhibition zone.
What is meant by inhibition zone?
A clear area around the plant extract in which it kills bacteria (bactericidal) or inhibits its growth
(bacteriostatic)
How to prepare the petri dish?
› Get a sterile petri dish.
› Add agar.
› Spread bacteria evenly over the surface of agar.
Why temperature 28°C?
High enough for proper enzyme activity of bacterial enzymes but still lower than core body
temperature of 37°C at which pathogenic bacteria can grow.
Why a vertically fixed lid?
To reduce entry of foreign bacteria & also to avoid creating anaerobic conditions that encourage
growth of anaerobic bacteria.

© Dr. Mohab Megahed

 61
How to handle bacteria safely in the lab?
› Flame the inoculating loop & the neck of the flask containing bacterial culture using a
bunsen burner.
› Use a harmless bacteria strain such as E.coli.
› Sterilize the laboratory bench before & after the investigation using cotton soaked in
ethanol.
› Incubate at temperature of 28°C to reduce entry of foreign bacteria & also to avoid
creating anaerobic conditions that encourage growth of anaerobic bacteria.
› Vertically fix the lid to reduce entry of foreign bacteria & also to avoid creating anaerobic
conditions that encourage growth of anaerobic bacteria.
› Autoclave all the used equipment before & after the investigation.
› Safe disposal of agar after the investigation.

Flask containing bacterial culture

Bunsen burner Inoculating loop

© Dr. Mohab Megahed

 66
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.
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

Practice: Calculate the magnification of the image below

Practice: If a plan drawing shows a leaf cross section, magnification 400x, and a nucleus
on it measures 0.5mm width, what is the actual nucleus width?

© Dr. Mohab Megahed

 67
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.
› 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.

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.

Cells stained by Iodine

Lignin Pectin DNA

© Dr. Mohab Megahed

 68
Core Practical: Using Light Microscope

Microscopes can be used to observe many types of specimens, from animal, plants and
microorganisms.

Laboratory prepared slides usually undergo:

› Dehydration
› Staining
› Slicing
› Embedded in wax to prevent sliding

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

© Dr. Mohab Megahed

 69
Plant Tissue preparation: (Stem, Root or Leaves)
› Lay the stem on a chopping board.
› 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)
› Ensure that you cut thin sections so that you become
able to see their components clearly.
› Place a drop of water onto the slide to house your
section.
› Place the stem section onto the slide.
› Use a mounted needle/forceps to lower the coverslip
onto it gently.
› Use absorbent tissue to mop up any leaked water at the
edges.
› Observe under microscope.

Observing samples of Animal/Plant cells:

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.

© Dr. Mohab Megahed

 70
Plan Drawings

› These drawings can be made when observing tissue or cell structures.

› To draw a tissue, we use the low or the medium power, to draw a cell we use the high
power.
› Always draw with a sharp HB pencil.
› Make a title explaining what the drawing is and what is the magnification used.
› Drawings should never include shading or colouring.
› Lines in a tissue drawing separate cell types.
› Draw to cover at least half the space provided and leave space for labels/annotations.
› Label tissue regions with sharp clear lines. Label lines shouldn’t intersect.
› Make it proportional to the sizes of structures in the real specimen.
› If italics needed, underline instead.

N.B. If you are drawing cells, only show structures visible inside cells such as cell wall, cell
membrane, cytoplasm & nucleus. Make sure to draw what you see not what you expected
to see!

© Dr. Mohab Megahed

 79
Graticule to make measurements
› Light/optical microscope enlarges in a linear fashion, so 100x magnification means that
the image is 100x wider & longer than the real object.
› The magnification is a product of the objective lens magnification and eyepiece
magnification.
› They eyepiece in a microscope can be fitted with a graticule – a transparent device acting
as a ruler.
› This means dimensions of what we see can be measured in eyepiece units (EPU).
› At different magnifications, EPUs represent different lengths, so we have to calibrate the
graticule for each objective lens.
› We use a stage graticule which is placed on the stage of the light microscope, to calibrate
the eyepiece graticule.
› This stage graticule is usually 1mm (1000um) long.

How to calibrate the EPUs:

› Place the eyepiece graticule into the 10x eyepiece of the microscope.
› Use the 4x objective lens and focus on the stage graticule.
› Use these 2 rulers aligned to work out how much distance one EPU represents at the
magnification of 40x. For example, if you found that 40 EPUs correspond to the stage
graticule which is 1000um in length, then 1 EPU = 25um.
› You can repeat this for any other objective lens.

Practice: If the smallest division in the stage micrometer is 10um. Calculate the size of the
smallest division of the eyepiece graticule.

N.B. Remember standard forms from your mathematics course.

© Dr. Mohab Megahed

 80
Microscopy Evaluation

Conclusions: Through microscopy we can conclude:

› The type of cell or tissue present under the microscope.
› The presence or absence of certain pathogens, tissue damage, tumour invasion or foreign
bodies.
› The size of certain features using graticules.

Errors:
› Errors in preparation of the slide:
• Contaminating the sample with fingers or other objects.
• Damage to the sample during preparation (by crushing for example)
• Improper use of stains
• Dirty slides/ lenses.
› Error in identifying histological features leading to misdiagnosis of pathogens or cancers.
› Error in size calculations or improper use of graticules.

Limitations:
› Bad preparation, staining or preservation of the specimens.
› Some features are difficult or impossible to stain so can’t be visualized.
› Poor resolution or magnification of the used microscope.
› Improper focus on the slides leading to blurring of the image.
› We only view a sample of a tissue not the whole tissue, so we can miss diagnosis of
abnormalities such as cancers or pathogens present.
› Some features appear differently in a transverse cut than longitudinal cut or in a 2D
image than in a 3D image. This might cause confusion.

Improvements:
› Careful preparation of the specimen.
› Examine more than one tissue sample.
› Double check calculations to ensure reliability.

© Dr. Mohab Megahed

 85
6) Investigating the effect of sucrose concentration on pollen tube growth
› Prepare pollen culture medium and add to it a known sucrose concentration.

› Add a drop of the solution you prepared to the center of a clean slide.

› To add pollen, use a low power microscope and knock pollen off the anthers of a flower
using a mounted needle.

› Observe under the microscope to note when pollen tubes start to grow,
this usually occurs after about 15 – 30 minutes depending on the used
plant species.
› At this point, use the eye piece and stage graticules to record the
pollen tube length every 3 minutes for half an hour.
› Repeat the same process with other slides using pollen from the same
anther and the same culture medium but with a range of different
sucrose concentrations.

© Dr. Mohab Megahed