1

Practical unit 4
(1) Investigating the effect of limiting factors on the rate of
photosynthesis
1-Effect of different light intensities.
*Independent variable: Different light intensities:
Place a lamp at distances 10,20,30,40,50 cm from the beaker.
*Dependent variable: Rate of photosynthesis:
-Volume of oxygen collected , (using a gas syringe or capillary tube)
-per unit time* , using a stop watch.
*Controlled variables: (for validity)
1) Same species* and mass* of pond weed (digital sensitive balance)
2) Same temperature  (electrostatic water bath.)
3) Same CO2 concentration (same mass of sodium hydrogen carbonate)
4) Same wavelength of light (use the same lamp, transparent, allows
white light) .
5) Same time interval( stop watch)
- Repeat the experiment 3 times at each* distance and calculate average.
( for reliability) . Draw a graph of the results and analyse statistically using
Spearman rank correlation test.
 Other methods of measuring the rate of photosynthesis:

-Volume of carbon dioxide absorbed / mass or concentration of glucose

formed, or increase in biomass
..per unit time*
 2

 Changing independent variable:

2) Different carbon dioxide concentrations:
Ex. : 5 different concentrations of sodium hydrogen carbonate.
3) Light wavelength: use light filters / lamps of different colours:
transparent (allows white light) , green, blue ,red ,yellow filters
at the same distance from the plant.
4) Temperature:
Five different temperatures :
10,20,30,40,50 °C using
thermostatic water baths.
Steps:
1- Place a piece of pond weed
in a beaker of water. Cover it
with a funnel connected to a
capillary tubing filled with water, and a syringe.
(Cover one side of the beaker with aluminum foil, so that light enters from
only one side ),
2- Add (0.5 g) sodium hydrogen carbonate to the water (source of carbon
dioxide)
3- Place a lamp 10 cm from the beaker.
4- Allow the pond weed to adjust / acclimatise for 5 minutes. When bubbles
of gas start to appear, start the stopwatch.
- Record the volume of gas (oxygen) produced over a fixed time interval.
5- Use the syringe to refill the capillary tubing with water  Repeat..
 3

Chromatography : Chloroplast pigments can be separated, identified by

this technique.
Pigments travel different distances up the filter paper/silica gel according to
- Their solubility in the solvent.
- Their molecular size.
Method: -
-Crush and grind plant tissue to extract the pigment, use a solvent ex
Propanone / alcohol, NOT WATER! (chlorophyll is NON POLAR!).
-Load it on filter paper on the origin (start line), and let it rise with the
solvent.--> (Pigment front)
-Repeat using the solvent only till it reaches the top -> (solvent front)
NB : Always use the same solvent , For Validity
RF : Ratio* of the distance* travelled by the pigment /( divided by)
Distance travelled by the solvent alone from the origin to the solvent front.
How to identify a pigment from:
1- Its RF look it up in a reference.
2- Its colour
 4

(2) Effect of Temperature on the development of organisms:

a) Brine Shrimp Hatch rate: Brine shrimps are small, salt water
crustaceans; the adults are about 8 mm in length. They are relatively easy
to keep in the laboratory and will produce
dormant egg cysts that hatch to produce young
1-Independent variable: different temperatures:
place a beaker of sea water in a separate water bath at
different temperatures 15,20,25,30, 35ºC, (why not higher?)
2-Add (40) brine shrimp eggs*. (Count using graph paper, and a magnifying lens).
3-Leave for 24 hours in the water bath, then count the number of eggs hatched.
( moving or swimming larvae)
4- Dependent variable: calculate the percentage of eggs hatched at each
𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑒𝑔𝑔𝑠 ℎ𝑎𝑡𝑐ℎ𝑒𝑑
temperature. Rate =
𝑇𝑖𝑚𝑒 𝑖𝑛 ℎ𝑜𝑢𝑟𝑠

5- Controlled variables: (same*)

- Species of shrimp (same source/batch
- Volume of water (measuring cylinder), salinity, pH (buffer solution),
O₂ concentration. - Light intensity.
6-Reliability: Replicate several times at each* temperature and get the average.
Plot a graph of the results ( what trend do you expect?)
7-Analyse statistically (Spearman rank correlation )

N.B. Experimental difficulty: counting the exact number of hatched eggs:

- To increase accuracy, take a sample of water using a pipette and examine
under the low power of light microscope.
 5

Alternative method: Percentage germination/Growth in plants.

1- Use 10 mustard /cress seeds placed on damp cotton wool in each petri
dish.

2- IV : Place each petri dish at different temperatures ( state 5 values)

- in a water bath / incubator..

3- Leave for 5 days, and water regularly so that they don’t dry out.

4- Count the number of seeds germinated / Dry mass or length of seedlings

at each temperature.

5- Use 3 petri dishes at each temperature (replications) to calculate the

mean.

 Controlled variables:

- Same species of seeds, and number of seeds in each dish.

- Same volume of water added to each dish.

- Same light intensity.

 6

Bacteria
-Bacteria reproduce asexually by binary fission (not mitosis),
quite rapidly, every 20 or 30 minutes under favorable conditions.
A colony is a cluster of bacterial cells arising from a single bacterial cell
by asexual reproduction.
Generation time: Time between two successive bacterial cell divisions.
It is short because bacterial cells are small, simple, and reproduce
asexually..
Stages:
1- Lag phase: slow increase in number as the bacteria is adjusting to
the available resources (activating genes, making enzymes)
2- Log (exponential) phase: Very rapid increase. A log scale is used on
the y axis to show a straight line (easier to analyse).
( log scale means that every number in the Y axis is to the power of 10!!)
- It indicates the number of LIVING bacterial cells!
3- Stationary phase: population size is stable,
Death rate equals division/reproduction rate. Limiting factors set in.
4- Death phase number falls as conditions continue to deteriorate,
nutrients running out, accumulation of waste products.
 Growth rate constant: (K)
can be calculated from the equation:
N0 = number of bacteria at the start.
Nt = number of bacteria at the end of
the time interval (t)
-Example: In 5 hours, the number of bacteria increased
from 5 x 104 to 9 x 106 , calculate K:
 7

 Conditions needed for bacterial growth and reproduction:

-Nutrients: source of Carbon, Nitrogen ( to make proteins)
-Oxygen ( for aerobic respiration),
-proper pH, warm temperature ( for enzyme activity).
 Types of culture media:
-Liquid: (nutrient broth)
-Solid: e.g. nutrient agar , Blood agar.
 Selective medium: Encourages the growth of one (or few) types of
bacteria, and not others ( useful in identifying organisms)
Gram +ve bacteria Gram -ve bacteria
Thicker cell wall Thinner cell wall
Contains teichoic acid Non
Stains blue/violet Stains red
 8

(3) Rate of growth of microorganisms in a liquid culture:

- Add certain mass of yeast to certain volume of glucose solution in a sterile*

flask. Stopper the flask with sterile* cotton wool*.
-Stir and incubate at 20°C for 10 hours.

 Measure Growth rate:

a) Optical density ( turbidity) ( indication of the density/ growth of yeast):

Spectrophotometer (connected to a data logger):
-Shine light through the culture tube , and record the light transmission
(remaining light that passes through it) using a light sensor .
-Take a sample of the culture every 30 minutes for 10 hours , to follow up
the growth rate* over time!!.). Light transmission would decrease over
time, indicating growth of yeast. (The more yeast cells present the higher
the turbidity the less the light transmitted).
- Compare it to a control ( identical glucose solution without yeast).
NB : This method measures both living as well as dead cells!!
 9

b) “Haemocytometer” and a light microscope:

This is a special microscope slide, with a grid of known volume , allowing
counting of stained cells ( Blood cells/bacterial/fungal cells)
- Take samples every 30 minutes and count the number of yeast cells.
(If yeast cells overlap dilute the yeast suspension!!).
4- Record the results in a table, draw a graph of the results.
5- Follow aseptic techniques all through.
NB: - Both living and dead cells can be counted.
- Can be used with an ( EXCLUSION DYE ) to show only living cells.
- The Cells on the edges can be either ignored , counted totally,
or 2 halves counted as one!
 10

3- Colony count: Spread the sample over solid agar, incubate for 24
hours, then count the number of colonies.
- Each single bacterial cell will divide many times to produce a single colony.
- Therefore the number of colonies should be equal to the original number of
bacteria.
NB : It represents only viable (living) cells!
Inaccuracy : colonies may clump together or overlap miscount.
Solution: - Dilution plating : Dilute the original bacteria or yeast
sample, serially and culturing it on agar plate each time, till you reach
countable colonies. Then: >
- Multiply : Number of colonies x reciprocal of the dilution sample
11
 12

Making serial dilutions:ex. With a dilution factor 2:

1- Add a certain volume from the original stock solution (ex 5 cm3)
2- Add EQUAL volume of distilled water ( also 5 cm3).
3-Total volume would be 10 cm3.
4- Do this successively ,,,Ex. obtained concentrations would be :
100%, 50%, 25%, 12.5%, 6,25%.....

𝒕𝒐𝒕𝒂𝒍 𝒗𝒐𝒍𝒖𝒎𝒆
 Dilution factor =
𝒗𝒐𝒍𝒖𝒎𝒆 𝒐𝒇 𝒔𝒕𝒐𝒄𝒌 𝒕𝒓𝒂𝒏𝒔𝒇𝒆𝒓𝒆𝒅
10
=2
5

What is the
Dilution
factor ?
 13

(4) The effect of different antibiotics on bacterial growth:

1. Use prepared sterile nutrient* agar plates seeded with a fixed mass of
bacterial culture of a known strain ( or prepare your own plates…see
below).
2. Pipette 0.1 cmᵌ of the antibiotic on equal sized sterile filter paper discs
and put on the nutrient agar using a sterile forceps (OR in holes (wells) in
the agar). The discs or holes should be equidistant and one with no
antibiotic (control) is done. (You can put 0.1 cmᵌ sterile water).
3. Tape the lid vertically: allows some air to enter.

(R = prevents anaerobic conditions which can encourage harmful bacteria to

grow.)
4. Incubate for 24 hours at 25°C

(R = higher temperature ex. 37°C is the human body temperature,, would

encourage harmful bacteria to grow.)
5. Observe without opening the lid (R = prevent spread of bacteria) .
Bacterial colonies appear on the agar except in the areas around the discs
(Clear zones). This is because the antibiotic DIFFUSES through the agar ,
killing the sensitive bacteria around the discs, or preventing its growth)
6. Measure the diameter of the clear area around the antibiotic ( inhibition
zone) using a ruler , or area using graph paper tracing.

The larger the area the more powerful is the antibacterial effect of the
antibiotic). Compare with the control !!
 14

 How to calculate the area:

-If circle: measure the diameter* using a ruler or Vernier caliper then TTr².
.(-If irregular, get 2 diameters, get their average and use it).
-Or use a graph paper tracing.
- Exclude the area of the disc / well.
7. Autoclave the whole instruments and plates before their disposal , and
clean the bench using a disinfectant.
8. Replicate the experiment several times at each…. and get the average .

Experiment can be used to compare:( independent variables ):

- Compare different types of antibiotics on one type of bacteria.
- Different concentrations of one type of antibiotic.
- Same antibiotic on different types of bacteria (use different plates with
different bacteria, in each add one antibiotic)
15
 16

Preparing sterile agar plates : Dissolve agar powder in boiling water in a

flask. Leave to cool, but not solidified. Add a fixed mass of bacterial culture
to the warm molten agar. This ensures that the bacteria are evenly
distributed in the agar. Now pour the agar with the inoculated bacteria into
sterile petri dishes. leave it to solidify before adding the discs.

Safety:
a) Reducing chances of contamination from bacteria in the air:
( for validity and safety)
1. All equipment and agar should be sterile , use autoclave for the
equipment and agar . Autoclave the plates before disposal.
2. Work near a Bunsen flame (or in a laminar flow chamber if available),
(to move away microorganisms in the air, preventing them from
contaminating the culture)
3. Clean the bench with disinfectant before and after work.
4. Wear sterile gloves, goggles, masks, hair cover and lab coats, tie hair
back.
5. Don’t incubate at human body temperature.
6. Tape the lid vertically at 4 places.
7. Don’t open the lid totally during pouring agar, or adding the extract.
Also don’t open lid after incubation.
B) others
-Care from burning while pouring hot molten agar (use heat resistant
gloves) .
17
 18

Methods of measuring abiotic factors:

Climate:
 Light intensity light meters.
 Temperature:
1)Glass thermometers: Too fragile,

2)Digital thermometers/temperature probes: Can measure at

different depths of soil / water, also some allow data logging  possible
to take continuous measurement over a long period of time .

 Humidity  Hygrometers.
 Rainfall  rain gauge.
Aquatic habitats: Take a sample of water!
 O2 concentration  Digital O2 sensor /probe.
 pH  pH meters/pH probe/ pH indicator .
Edaphic factors(Soil) Take a soil sample!!
 Soil pH pH meters /pH probe/ pH indicator .
 Moisture content Weigh the soil sample (sensitive balance) ,
then dry it in an oven/incubator at 60oC, till a constant mass is
reached, and calculating % mass loss.
𝑴𝒂𝒔𝒔 𝒍𝒐𝒔𝒕
𝒙𝟏𝟎𝟎
𝒐𝒓𝒊𝒈𝒊𝒏𝒂𝒍 𝒎𝒂𝒔𝒔
 Mineral ion content : chemical kits.
 Soil type and structure: Measure size of soil particle, extent of
sand and clay/humus content.
 Air spaces: measure water drainage rate.
 Soil and water temperature: IN SITU!, Not a sample!
Digital thermometers/temperature probes inserted into the soil.
Topography (Earth's features)
 Slope  clinometers. Aspect  compass

Sampling: See theoretical notes Topic 5.

19