EMERGING ISSUES IN BIOLOGY

SBU 1043

LABORATORY MANUAL

DEPARTMENT OF BIOLOGY

FACULTY OF SCIENCE AND MATHEMATICS

 TABLE OF CONTENT

NO. EXPERIMENT PAGE

1 Experiment 1: Biological Instruments 3

2 Experiment 2: Isolation of microorganism 4
3 Experiment 3: Methane production from food waste and garbage 6
4 Experiment 4: Observing the influence of acid rain on seeds 9
germination and plant growth
5 Experiment 5: Genetics intervention in life (1) 13
6 Experiment 6: Genetics intervention in life (2) 15
7 Experiment 7: Conservation Biology 18

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Experiment 1: Biological Instruments

Objectives:
1) Identify selected biological instruments used in the biological research
2) Understand the function and application of the selected biological instruments

Materials:

 Ion Chromatography
 Inverted Fluorescence Microscope
 Spectrophotometer UV-Vis
 Deionized water
 Freeze Dryer
 -80⁰C ultra-deep freezer

Methods:

In this practical session, you will be introduced to several instruments available in Biology
Department by the lab assistant. While listening to the explanation, write down the important
features/characteristics as well as functions of the instruments. Pictures of each instrument
should also be included in the lab report.

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Experiment 2: Isolation of microorganisms

Objectives:
1) To isolate bacteria populations from a mixed culture
2) To differentiate microbial population on hands that has been washed in either Dettol
or 70% alcohol
3) To isolate microorganism from water sample

A) Isolation of bacteria from mixed population

Materials:
Nutrient agar
Mixed culture in broth
Wire loop
Alcohol
Dettol

Methods:
1. Aseptically remove a wire loop of mixed culture from the broth and streak it on the
nutrient agar plate using the streak plate method. Shake the bottle containing
mixed culture broth before use.
2. For isolation of microorganism from thumb, divide the plate into 3 parts, A, B and
C. Place unwashed thumbprint on part A. Student B will wash his/her hand with
70% alcohol while student C wash his hand with dettol. Place your thumbprints on
part B and C.
3. Incubate all nutrient agar plates at 37°C overnight.

B) Isolation of bacteria from water sample

Materials:
Vacuum pump with sterile filter apparatus
Membrane filter disk (0.22µm)
Whatman No. 1 filter paper
Nutrient agar
Pond water sample
Sterile water
Pipette and tips

Methods:
1. Filter 200ml of pond water sample using Whatman no. 1 filter paper to remove
unwanted debris.
2. Assemble the membrane filtering apparatus:
a. Aseptically insert the filter holder base into the neck of a 1L side arm flask.
b. With a flamed forcep place a sterile membrane filter disc grid side up, on the
filter holder base.
c. Place the filter funnel on top of the membrane filter disc and secure it to the
base with the clamp.
d. Attach the rubber hose to a pump.
3. 100ml of water sample is poured into assembled funnel, utilizing vacuum.
4. Rinse the inner sides of the funnel with 20ml of sterile water.
5. Remove filter disc carefully with sterile forceps and place on nutrient agar with the

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 grid side up.
6. Incubate at 30oC for 24 hours.
7. After incubation, count the colonies on the disc and transfer isolated colonies to a
new nutrient agar.
8. Incubate at 30oC for 24 hours to obtain pure culture.

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Experiment 3: Methane production from food waste and garbage

Objectives:
1) Learn about methanogens
2) Understand the chemistry behind the production of methane from food waste
3) Identify the gas or gases produced during the reaction

Background:

Biogas is 60-80% methane and is created by a process termed anaerobic digestion, leaving
behind a nutrient- rich substance termed digestate. Anaerobic digestion is carried out by a
range of bacteria in the absence of oxygen. Initially carbon dioxide is produced aerobically
by the decomposing organic matter until an anaerobic environment is created. After the
initial digestion a group of bacteria known as methanogens convert the feedstock into
methane and carbon dioxide.

Anaerobic digestion has a number of environmental benefits including production of

‘green’ energy and natural fertilizers. The process of converting organic feedstock into
biogas can can serve as a substitute for fossil fuels and artificial fertilizers, reducing the
amount of greenhouse gases released into the atmosphere. The problems associated with
waste disposal are also alleviated by the generation of useful products and decreased
release of the potent greenhouse gas, methane, from landfill sites.

Methanogens are obligate anaerobes that cannot grow in the presence of oxygen. They use
CO2 as the final electron acceptor and Hydrogen as a source of electrons. The reduction of
CO2 produces methane gas as a byproduct of cellular metabolism. Methanogens are
abundant in swamps and sludge water. They play an important role in biomass degradation
and CO2 consumption.

CO2 + 4 H2 CH4 + 2 H2O

Methane is a colorless and odorless gas and the main component of natural gas (over 75%).
Methane is combustible and is a major source of fuel for heat, cooking and electricity
production. Natural gas is a fossil fuel that is not renewable. Although there are still huge
deposits of natural gas in the US, extracting them is expensive and is environmentally
destructive, and finding alternative renewable sources of methane (biogas) is a high priority.

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Methane burning consumes oxygen and produces carbon dioxide and water.

CH4 + 2O2 CO2 + 2 H2O

Methanogens are abundant in anaerobic freshwater such as swamps, the stomachs of

ruminants, and sewage sludge. Methane is produced naturally by the anaerobic
decomposition of organic matter. (Think swamp gas.) Finding new organisms that can
efficiently convert biomass into methane is an active area of research.

Using food waste and garbage to produce methane appears to be an ideal way to use
biology to solve two societal problems - to produce fuel while getting rid of garbage. In this
experiment food waste is incubated with sludge water in a tightly sealed container. Aerobic
decomposition occurs first and CO2 is produced while Oxygen is consumed. Once the
oxygen has been depleted the methanogenic bacteria start growing and consume the CO2
to produce methane.

Therefore, one should expect an initial increase in CO2 production followed by a decreasein
CO2 and a steady increase in methane production. The biogas obtained at the end ofthe
reaction will be a mixture of CO2 and CH4 . The CO2 can be monitored using aCO2
detection probe. The methane can be identified by burning it. The biogas will be collected in
a balloon and burned under the hood.

Materials:

Glass bottles with stoppers (we will use 125 ml serum bottles.) You can also use plastic
bottles with outlets
Needles (18 to 22 gage)
Blender
Tap Water
Sludge or swamp water
Graduate cylinders
Tubing
Balloon

Procedure:
• Fill half of the blender with food waste from your kitchen.
• Add water

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• Turn on the blender. Add enough water to obtain a mixture that has the consistency
of a hearty soup: thinner than chowder but thicker than bisque.
• Add 40 ml of the waste mixture two each of 250 ml BOD shaped bottles.
• Label one flask: Sludge and the other water (control)
• Use a graduate cylinder to add 10 ml of bacterial sludge (now 20% by volume of the
total) from Deer Island or other sources to the flask labeled sludge.
• Add the same amount of tap water or autoclaved sludge to the other control flask.
• Check the pH. It should be around 7. If necessary add some base to increase pH to 7.
• Seal the bottle with a blue stopper (Wet the stopper first to make it easier to push into the
bottle).
• Next, take a new 1-ml insulin syringe and remove the plunger, cut the top of the
barrel to remove the flaps. Then screw the bottom of the syringe into the needle.
• Attach the top of the syringe barrel to a length of soft (tygon) tubing – it should be
airtight. If necessary, use electrical tape to seal the connection.
• Attach a balloon to the end of the tubing OR bubble gas into a collecting tube over
water
• When the balloon-tubing-syringe-needle apparatus is complete, you may then
carefully insert a needle into the stopper and push through until it comes out the other
side of the blue stopper.
• Incubate the set-up at 37ºC.
• Measure CO2 produced using the method you have chosen.
• At the end of the experiment check the pH of the solution.
• Add bleach to liquid waste and discard
• Throw solid waste into burn box.
If time permits you can dry and weight the solid waste

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Experiment 4: Observing the influence of acid rain on seeds germination and plant
growth

Objectives:
1. Justify the effect of acid rain on seeds germination and plant growth
2. Explain how acid rain affect plant’s growth
3. Identify plant’s tolerance to acidity level of acid rain

Introduction

Industries and motor vehicles produce gaseous oxides of nitrogen and sulfur. For example
nitrogen and oxygen in the air can combine under high-temperature engine conditions to
produce nitrogen dioxide (NO2). The equation for the reaction is:

N2 + 202 --> 2NO2.

Sulfides in fuels can combine with oxygen to make sulfur dioxide (SO2) and sulfur trioxide
(SO3). Such oxides combine with water in the atmosphere to make acids. For example,
nitrogen dioxide and sulfur trioxide combine with water. They form nitric acid (HNO3) and
sulfuric acid (H2SO4), respectively. The equations for these reactions are:

 3NO2 + H20 ---> 2HNO3 + NO

 SO3 + H20 ---> H2SO4.

The presence of these acids causes rain to be acidic. Acid rain damages trees, crops, and
buildings. It can make lakes so acidic that fish cannot survive. In this experiment, students
are required to stimulate the effect of acid rain on plant by comparing the effect of varying
levels of acidity on seeds germination and plant growth.

Materials
pH meter
Weighing balance
500mL beakers
500mL glass bottles
1M H2SO4
4” plastic plant pots/small planting bag
Commercial potting mix

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Corn seeds
Spray bottles

Procedure
Planting seeds and preparation of acid rain solution
1. Fill 12 plastic plant pots with soil until the pot is ¾ full
2. Label the pots as the following:
a. Pot 1: Control (triplicate a,b and c)
b. Pot 2: Acid rain A (triplicate a,b and c)
c. Pot 3: Acid rain B (triplicate a,b and c)
d. Pot 4: Acid rain C (triplicate a,b and c)
3. Plant six corn seeds in each pot, approximately 15mm deep
4. In the mean time, prepare the following acid rain solutions by mixing tap water with 1M
H2SO4
a. Fill up a beaker with 500mL tap water.
b. Adjust the pH of the tap water to pH 5 (acid rain solution A), pH 3 (acid rain
solution B) and pH 1 (acid rain solution C).
c. Transfer each solution to a labeled spray bottle.
5. Water each pot with:
a. Pot 1: Tap water
b. Pot 2: Acid rain solution A
c. Pot 3: Acid rain solution B
d. Pot 4: Acid rain solution C
Note: Use spray bottle to water all pots with approximate volume to wet the
seeds and soil. Note the planting day as day 0
6. Place pots on plastic trays and place trays on lab’s bench
Treatment and observations
1. Water each pot with the respective acid rain solutions (use tap water for pot 1) on day 1,
2, 4, 6
2. Observe the pots and determine for each the number of plants that have either
germinated or have become a seedling and record this number in Table 1.
3. Observe the pots and record for each a qualitative description of plant including leaf
color, lesions and spotting on leafs and stems, presence of dead leafs, and any other
signs of damage.
4. On day 8 (or next laboratory meeting), harvest plants from each pot and determine the
following:
a. The final qualitative description of plants for each treatment pot

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 b. The average plant height (in cm) for each treatment pot
c. The average plant mass (in gram) for each treatment pot

Table 1: Germination, growth and physiological condition of corn plants watered with
simulated acid rain (pH 1, pH 3, pH 5)
Pot Pot 1a,b,c Pot 2a,b,c Pot 3a,b,c Pot 4a,b,c
Watered with: Tap water Acid rain A Acid rain B Acid rain C
(pH 5) (pH 3) (pH 1)
Day
0 # Germinated: # Germinated: # Germinated: # Germinated:
.
.
.
.
6 Description: Description: Description: Description:

Final
physiological
condition

The average
plant height
(cm)

The average
plant mass
(gram)

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Questions
1. Prepare graphs illustrating the seeds germination profile for each treatment pot
2. What type of relationship that exists between seed germination and pH?
3. Describe the observed pattern between seedling growth and damage and pH?
4. Some rain has been found to be as acidic as pH 2.8. Given what you have observed,
what is the likely effect on seed germination and plant reproduction?
5. How acid rain can affect plant growth? Please provide your explanation in relation to
plant physiology and plant nutrients.

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Experiment 5: Genetics intervention in life (1)

Objective:
To determine the effects of ultraviolet light and magnetic field on seed germination and the
growth of mug beans.

Materials and Methods:

mug bean seeds
plastic petri dish
cotton
water
magnetic bars
lamina flow

Procedures:
1. First soaked the mug bean seeds in water for 24 hours.
2. Spread the cotton into a plastic petri dish. Moist the cotton by spraying water on the
cotton. Then placed approximately 10 seeds on wet cotton. Repeat this step 9 times
to produce replicates for this setup.
3. Place 6 petri dishes under ultraviolet light in a lamina flow for 30 minutes. After 30
minutes, turn off the UV light and remove 2 petri dishes and label them as 30 min
treatment. Turn on the UV lights again and let the remaining 4 petri dishes under the
UV light for 1 hour and remove the petri dish from the lamina flow, and label
accordingly. Repeat this procedure for the remaining 2 petri dishes under UV light
treatment for 2 hours.
4. Place two untreated petri dishes near two magnetic bars according to the
arrangement shown in the figure below. Leave the experiment setup in this
arrangement for three days.
5. Standby two petri dishes without any treatment to serve as the control.
6. Place all the 10 petri dishes setup near light sources for 5 days.
7. Record your result by measuring the length of newly emerged mugbean stem (in
milimeter) for day 1, 2, 3, 4 and 5.

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Recording results:

1. Typical results for this experiment are listed in the table and photographs below.

Treatme Total beans germinated (out of 10) Com

nts Da Day Day Day Day ment
y1 2 3 4 5 s
UV 1
hour
UV 1.5
hours
UV 2
hours
Magneti
c bars
Control

2. Include pictures of sprouting mung beans where necessary.

3. Construct a histogram with display beginning with scales of zero in both x-coordinate
and y-coordinate.

Petri dish
Magnetic bar

S N S
Figure 1 N S N

Cautious: Please switch off the UV light whenever you are working under the lamina flow.
Exposure to UV lights is harmful to skin, eyes and immune system.

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Experiment 6: Genetic Intervention in Life (2)

Objective:
To determine the effects of ultraviolet light and magnetic field on the growth of E.coli.

Materials:
Bacteria and fungi broth culture
LB agar plate
Bunsen burner
Inoculation loop
Latex gloves
Paraflim

Streaking:

1. Flame the loop to sterilize it and let cool.

2. Dip the loop into the broth culture containing the mixture of bacteria
3. Position the plate so that the spot of inoculum is nearest the hand not holding the
loop (the opposite hand).
4. Lift the plate lid with the opposite hand.
5. Move the loop back and forth across the spot and then gradually continue toward the
center of the plate as you sweep back and forth. Use a very gentle and even
pressure (refer to Figure 2).
6. When creating each phase, do not worry about keeping each pass across the plate
separate from previous ones.
7. When about 30% of the plate has been covered by the first streaking phase, remove
the loop and flame sterilize it.
8. Repeat the above procedure for the second phase, but this time pick up some
inoculum by crossing into the first phase 2 to 3 times and then not passing into it
again.
9. Repeat as necessary for the third and fourth phases. After streaking the plate, flame
sterilize the loop before setting it down.
10. Repeat step 1 to 9 for 9 times to generate replicates.

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Figure 2: Streaking patterns. Arabic numbers indicate phases.

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Treatments:
1. Place 6 cultured petri dishes without cap under ultraviolet light in a lamina flow for
30 minutes. After 30 minutes, turn off the UV light and remove 2 petri dishes and
label them as 30 min treatment. Turn on the UV lights again and let the remaining 4
petri dishes under the UV light for 1 hour and remove the petri dish from the lamina
flow, and label accordingly. Repeat this procedure for the remaining 2 petri dishes
under UV light treatment for 2 hours. After treatments are done, cover of petri dish
with its top cover and seal the sides with parafilm.
2. Place two untreated sealed cultured petri dishes near two magnetic bars according
to the arrangement shown in the figure 1. Leave the experiment setup in this
arrangement for two hours. After 2 hours transfer them into an incubator (See step
4).
3. Standby two sealed cultured petri dishes without any treatment to serve as the
control.
4. Place all the 10 sealed cultured petri dishes setup inside the incubator at 37°C with
bottom facing upward.
5. Record your observation after one, two and three days.

Recording results:

1. Provide a suitable table to record your observation.

2. Include pictures of bacterial growth where necessary.
3. Construct a histogram with display beginning with scales of zero in both x-coordinate
and y-coordinate.

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Experiment 7: Conservation Biology

Objective:
To determine the amount of salt mung beans can tolerate before failing to germinate.

Materials and Methods:

Teaspoon
Measuring cup (250ml or larger)
Accurate kitchen scales or weighing balance (shared)
Petri dish
Table salt (NaCl)
Distilled water
Mung beans (dry)
Measuring jug or beaker
Tweezers (shared)
Eyedropper or plastic dropper for adding salt solution (shared)

A. Preparing salt solutions

Procedures:
1. Measure salt required into cup on scales (see table below for amount to use).
2. Add salt to measured volume of distilled water and stir. Repeat for each of the
concentrations required as per table below.

Amount of salt to add (in grams) to demineralised water (in millilitres) for various salt
solutions:
Salt concentration (c) Grams of salt to add (s)
(by weight*) 250ml 500ml
Tap water 0g 0g
0% 0g 0g
0.25% 0.6g 1.3g
0.5% 1.3g 2.5g
0.75% 1.9g 3.8g
1.0% 2.5g 5.1g
1.5% 3.8g 7.6g
3.5% (equiv. to 9.1g 18.1g
seawater)

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salt to add = concentration × weight of water ÷ ( 1 – concentration) s = c W / ( 1 – c ) [* Note:
1 ml of water weighs 1 gram]

B. Preparing ‘Petri dishes’

Procedures:
1. Prepare 8 plastic petri dishes.
2. At the bottom of each petri dish, add label to petri dish by using a permanent marker
pen (see below for suggested label details).

Mung Bean Germination Experiment Salt (NaCl)

Concentration : __________________
Date : __________________
Prepared by : __________________

3. Use appropriate amount of cotton wool (enough to cover the whole surface of petri
dish) and place it on the surface of each petri dish.
4. Use an eyedropper or pipette to moisten paper towel – drain off any excess water.
5. Repeat step 2 to 4 using different concentration of salt solution.

C. Adding Mung Bean seeds

Procedure:
1. Carefully place dry mung beans on moist cotton wool with tweezers.
2. Space dry mung beans evenly apart on paper towel.
3. Store in a well-lit area but away from direct sunlight to avoid towel drying out –
observe daily for next 4 to 7 days.

Note: Move dishes carefully to ensure the beans do roll around – separate any beans that
have rolled into each other after moving the container. Mung beans will sprout overnight on
moist cotton wool and do not necessarily need to be stored in a dark place.

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D. Recording results

4. Typical results for this experiment are listed in the table and photographs below.

Salt Total beans germinated (out of 20) Commen

Concentrati Da Da Da Da Da ts
on y1 y2 y3 y4 y5
Tap water
0%
0.25%
0.5%
0.75%
1.0%
1.5%
3.5%

5. Include pictures of sprouting mung beans where necessary.

6. Construct a histogram with display beginning with scales of zero in both x-coordinate
and y-coordinate.

E. Discussion
Discuss the objectives of the Mung Bean Germination experiment – to determine the amount
of salt mung beans can tolerate before failing to germinate. Discussion should be answering
these leading questions:
1. How could we find out how much salt mung beans can tolerate?
2. How much salt should we use?
3. Can we use plain old tap water?
4. Does it have any salt already in it?

F. Conclusion
This section should discuss the results and important observations from the experiment.

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