My Classroom Material 80 - BIOLOGY JUNCTION

Sample 3 Bioremediation

Bioremediation of Oil

Introduction

Public awareness of oil spills has increased over the years as more publicity has been focusing on this subject and the massive harm it does to our environment. The problem of oil spillage has increased as well, and recent studies suggest that 5-10 million tons of oil are spilled into the ocean annually. There are six major sources of spilled oil. The first is cargo tanker washings at sea. These oil tankers use seawater to stabilize their craft after discharging their oil, and then, the contaminated water is discharged into the sea when the tanker is refilled. Another source is waste oil pumping at sea which contributes 500,000 tons of oil annually. Also in-port oil losses are caused by collisions at port and the procedures used during loading and unloading. This contributes over one million tons annually. Exploration losses occur because of accidental damage to offshore drilling rigs and the blowout of wells. The last source is motor oil. It is assumed that over 2 million tons of unaccounted for motor oil reach coastal waters every year.

The damage oil spills can create is often underestimated. A single gallon of oil can actually spread over four acres of ocean. Oil spills can have devastating effects on marine life. Oil that remains after evaporation goes through an emulsification process that creates a highly viscous, sticky material. This material becomes thick enough to sink to the bottom of the ocean. It sticks to anything it comes in counter with including marine wildlife. Oil left after emulsification is broken down by microorganisms and photo-oxidation. After 3 months only 15% of the original oil volume remains and it forms tarry clots that float to shore. However, if the oil spill is extensive or occurred close to the shore line, the oil would not have sufficient time for emulsification and evaporation so the oil would create a viscous film of oil over any solid surface that comes in contact with the oil.

The short-term effects of these types of oil spills include four main areas of the environment. One effect is reduced light transmission. Light intensity under an oil spill can be reduced by up to 90%. This thwarts marine plant and protist growth because they can no longer use photosynthesis. Another effect is reduced amounts of dissolved oxygen in the water. Since the surface of the water is covered by the filmy oil, the water can no longer uptake oxygen. Oil spills also affect marine birds. Oil covered birds can be drowned or become handicapped. It also reduces their ability to fly and float on water, and their feathers no longer serve as insulation leaving them to die from exposure. The last area of short-term effect is the toxic effect of oil on the marine environment. Compounds such as benzene, toluene, xylene, naphthalene, and phenanthrene are toxic to man and marine life. Within only days oil spills can do massive damage to the marine life.

In addition to short-term effects, there are also long-lasting more serious effects of oil spills. The chemicals found in oil can mimic the chemical messengers found in oceanic water. This can deceive many marine animals that depend on these messengers to find food, escape from predators, or locating habitats suitable for reproduction. More stable oil components can be consumed by smaller species and passed on to animals higher up on the food chain, including humans. Oil can also serve as a concentration medium for other toxins like pesticide allowing them to reach humans and marine animals in a more potent form.

To reduce these effects, clean-up efforts must be fast and effective. There are four main mechanical methods used when cleaning an oil spill. Skimmers can be used to pull a thin layer of water off the surface and then separating the oil and the water. The oil can then be disposed of and the water returned to the ocean; however, this is only effective in calm waters. Booms or barriers can be used to contain the oil spill that will later be skimmed so it does not spread any further. Absorbent materials such as cloth, straw, powdered clay, and pine bark are spread over the spill surface. Chemical dispersants, detergents, and solvents can also be used, but these substances are toxic to the open water marine life and shore-dwelling organisms. The last mechanical method is burning the oil off the surface. This method was ineffective because oil is not completely combustible and it left carcinogens and thick toxic smoke over the area.

Bioremediation is the newest method of oil spill clean up and far more effective than any of the mechanical methods used. Mechanical methods are costly and can only remove about 10-30% of the contamination. Bioremediation makes use of indigenous oil-consuming microorganisms, called petrophiles, by enhancing and fertilizing them in their natural habitats. This microbial clean-up method cleans the oil as well as a number of other harmful pollutants and is perhaps the best, most environmentally safe process used today.

Petrophiles are very unique organisms that can naturally degrade large hydrocarbons and utilize them as a food source. This makes them singularly qualified for cleaning oil spills and even tanker bottoms containing oil residue. In bioremediation several different types of these microorganisms are used on the oil slick. They are dispersed by boats or aircraft and may be mixed with nutrients. The oil slick begins to show visible signs of degradation after only days. The microbes can then be assimilated up through the food chain leaving having no adverse effects on the marine environment.

Hydrocarbon degradation is an aerobic process. It uses oxidation to break down the large hydrocarbons in exposed petroleum into acetates and then eventually carbon dioxide. This final and complete oxidation into carbon dioxide is performed by the Krebs cycle, which creates components needed for the synthesis of amino acids and other compounds the microorganism may need.

Hypothesis

The oil in all three investigations will be visibly reduced by both oil-consuming microorganisms and this process can be measured by turbidity and the appearance of the oil on the surface. The sand in Investigation #3 may hinder or slow the degradation process.

Materials

Investigation #1: Visual Determination of Oil Degradation

The materials needed for Investigation #1 were 2 test tubes with caps, 60mL of distilled water, a density indicator strip, 1 test tube rack, 3 sterile pipettes, 8-10 drops of refined oil, .5 mL of the Pseudomonas culture, and .5 mL of the Penicillium culture.

Investigation #2: Simulated Oil Spill Clean Up in Water

The materials needed for Investigation #2 were 2 clear jars with caps, 400 mL of distilled water, a density indicator strip, 2g nutrient fertilizer, 30-40 drops of refined oil, 3 sterile pipettes, 1.25 mL of the Pseudomonas culture, and 1.25 mL of the Penicillium culture.

Investigation #3: Simulated Oil Spill along the Shore

The materials needed for Investigation #3 were 2 petri plates, 60 mL of distilled water, sand, 2 g nutrient fertilizer, 3 sterile pipettes, 30-40 drops of refined oil, 1.25 mL of the Pseudomonas culture, and 1.25 mL of the Penicillium culture.

Methods

Investigation #1: Visual Determination of Oil Degradation

Two tubes were labeled as follows: Tube #1: Pseudomonas species, Tube #2 Penicillium species. 5 mL of distilled water were added to each tube. Next, 4-5 drops of oil were added to each tube and allowed to form a thin layer over the water surface. Notes were taken on the oil’s general appearance. With a sterile pipette, Tube #1 was inoculated with 0.5 mL of the Pseudomonas culture, and Tube #2 was inoculated with 0.5 mL of the Penicillium culture. The cap was placed on both tubes and each was inverted several times allowing the microorganisms to mix with the oil. With caps loosened one-half turn, the tubes were incubated at 30° C. Each tube was observed and inverted every 24 hours for 4 days and each observation was recorded in the appropriate table.

Investigation #2: Simulated Oil Spill Clean Up in Water

The two jars were labeled as follows: Jar #1 Pseudomonas species, Jar #2 Penicillium species. Both jars were filled half way with distilled water. 15-20 drops of oil were added to each jar and allowed to spread over the entire water surface. An initial observation was taken of the oil appearance. Fertilizer was sprinkled over the entire oil layer in both jars. Using sterile pipettes, Jar #1 was inoculated with 1.25 mL of the Pseudomonas culture, and Jar #2 was inoculated with 1.25 mL of the Penicillium culture. The jars were then incubated at 30° C for four days with the caps loosened one-half turn. Observations were taken every 24 hours for the four days and a disposable pipette was used to blow bubbles into the culture after each observation.

Investigation #3: Simulated Oil Spill along the Shore

The lids of the petri plates were labeled as follows: Petri dish #1- Pseudomonas species, Petri dish #2- Penicillium culture. A layer of sand was spread 4-5 mm thick in each plate. The sand was then moistened with distilled water in both dishes. 15-20 drops of oil were added to each dish, and then fertilizer was sprinkled over the entire oil surface. Using sterile pipettes, Petri dish #1 was inoculated with 1.25 mL of the Pseudomonas culture, and Petri dish #2 was inoculated with 1.25 mL of the Penicillium culture. The petri dishes were then incubated at 30° C for four days. The plates were observed every 24 hours and recorded in the appropriate tables.

Results

Investigation #1: Visual Determination of Oil Degradation

Observations: Pseudomonas Species Application

Table 1

	General appearance characteristics of oil	Color of oil	Turbidity of water
Day 0	The oil is very slick and shiny in appearance. It covers the entire surface of the water. It is very transparent.	Transparent light amber	0
Day 1	Oil beginning to degrade. More oil in a ring around the tube. Very foamy, especially in the center. Oil is slightly darker and less transparent.	Light golden-yellow	0
Day 2	Oil continues degradation. It is still in a ring around the outside, however clear water is beginning to show within the center. The oil still has foam around it and appears cloudy	Pale cloudy yellow	1
Day 3	Less oil appears in the ring around the edge of the tube. The water in the center is clearer. Foam still present and the water is less cloudy.	Pale cloudy yellow	0
Day 4	The oil appears to be dispersing and running down the vile. The water and oil are cloudier and the oil is darker in color.	Cloudy amber	1

Observations: Penicillium Species Application

Table 2

	General appearance characteristics of oil	Color of oil	Turbidity of water
Day 0	The oil is very slick and shiny in appearance. It covers the entire surface of the water. It is very transparent.	Transparent light amber	0
Day 1	Oil beginning to degrade. More oil in a ring around the tube. Very foamy, especially in the center. Oil is slightly darker and less transparent.	Light golden-yellow	0
Day 2	The oil is more concentrated in the ring around the tube. Slightly less oil then Day 1. Appears to be continuing degradation.	Cloudy yellow	0
Day 3	Clear water is beginning to appear in the center of the water surface. Oil is more cloudy and darker.	Cloudy golden-yellow	0
Day 4	Less oil appears in the center as degradation continues. Oil still cloudy and dark.	Cloudy golden-yellow	0

Investigation #2: Simulated Oil Spill Clean Up in Water

Observations: Pseudomonas Species Application

Table 3

	General appearance characteristics of oil	Color of oil	Turbidity of water
Day 0	Oil is only present in a slick transparent layer across the surface of the water.	Amber	0
Day 1	Oil formed a ring around the outside of the jar and foam appears on the oil. Water is darker and oil is cloudier.	Cloudy amber	2
Day 2	The oil is not as slick on the surface and holes of clear water are beginning to appear on the surface as the oil breaks apart. The oil is cloudy and darker in color.	Pale orange-yellow	5
Day 3	Oil is increasingly cloudy and darker as more oil breaks up. Surface is no longer smooth and very foamy.	Cloudy orange-yellow	5
Day 4	Oil is in small clumps on the surface, but still forms a ring around the outside.	Cloudy dark yellow orange	5

Observations: Penicillium Species Application

Table 4

	General appearance characteristics of oil	Color of oil	Turbidity of water
Day 0	Oil is only present in a slick transparent layer across the surface of the water.	Amber	0
Day 1	Oil formed a ring around the outside of the jar and foam appears on the oil. Water is darker and oil is cloudier.	Cloudy amber	1
Day 2	The oil is not as slick on the surface and holes of clear water are beginning to appear on the surface as the oil breaks apart. The oil is cloudy and darker in color.	Pale orange-yellow	1
Day 3	Oil is thicker around the walls of the jar and oil appears to be broken up into sections and no longer smooth across the surface.	Cloudy orange	1
Day 4	There appears to be less oil on the surface and it is increasingly dark and cloudy.	Cloudy dark orange	5

Investigation #3: Simulated Oil Spill along the Shore

Observations: Pseudomonas Species Application

Table 5

	General appearance characteristics of oil	Color of oil	Turbidity of water
Day 0	The sand does not allow the oil to form a smooth layer across the water and oil appears in small transparent clumps across the surface.	Light amber	0
Day 1	Oil is spread more evenly across the surface. Some oil has seeped into the sand. Oil is not slick across the surface.	Light amber	0
Day 2	There is less oil on the surface and it appears to be slightly darker in color. It again appears in smaller sections across the surface.	Amber	0
Day 3	Most of the oil has seeped into the sand as the water has evaporated. There is less oil on the surface and the visible oil is still darker in color.	Amber	0
Day 4	Most of the oil has seeped into the sand as the water has evaporated. There is less oil on the surface and the visible oil is still darker in color.	Amber	0

Observations: Penicillium Species Application

Table 6

	General appearance characteristics of oil	Color of oil	Turbidity of water
Day 0	The sand does not allow the oil to form a smooth layer across the water and oil appears in small transparent clumps across the surface.	Light amber	0
Day 1	Spread more evenly across the surface, but still in small yellow clumps across the sand.	Light amber	0
Day 2	Oil is slightly cloudy and spread in clumps over the surface. Appears to be degraded slightly in the oil is darker.	Amber	1
Day 3	Oil formed a ring around the petri dish wall and the looks more cloudy and dark.	Amber	1
Day 4	Most of the oil has seeped into the sand as the water has evaporated. There is less oil on the surface and the visible oil is still darker in color.	Amber	0

Investigation #1: Visual Determination of Oil Degradation Questions

Describe the physical characteristics and appearance of oil on Day 0.

The oil on Day 0 had not yet been degraded in any way. It was a transparent light amber that stayed completely separate from the water. It formed a thin, slick layer over the water surface.

Describe any changes in the physical characteristics and appearance of the oil on Day 1 and beyond, and discuss possible causes for such changes.

The oil began to get darker and lose its slick qualities. It no longer covered the entire surface of the water and was separated into sections.

Is there a difference in the rate of oil degradation between the bacterial and fungal cultures?

The bacterial culture degraded the oil slightly faster possibly because bacteria can reproduce at a faster rate asexually.

Can you identify which is the bacteria and which is the fungus on agar slants? Describe the growth and appearance characteristics of both types of microbes. Can you think of any advantages in using bacteria over fungus to degrade oil?

The fungal colonies are larger than that of bacteria, but the bacteria was spread over a larger area and had more numerous smaller colonies. Bacteria would be more advantageous because it could spread over a larger area faster because it reproduces quickly and abundantly.

What does an increase in turbidity indicate?

An increase in turbidity indicates the break down of oil and the growth of colonies.

What is the turbidity level of your cultures after four days of incubation? How long do you think your cultures will continue to grow?

The turbidity of our culture was 1 after four days of incubation. The colonies will most likely grow until all the available oil has been degraded.

What is the limiting growth factor in your test tubes?

The amount of oil available limits growth in our test tubes and also possibly the amount of surface area.

Suggest optimum conditions that promote growth of bacteria and fungi. Suggest optimum conditions to culture bacteria and fungi.

Bacteria and fungi grow in any damp, warm area where there is any kind of available food source however optimum growth conditions are probably at about 30° C.

Investigation #2: Simulated Oil Spill Clean Up in Water

Describe what happens to oil after several days of microbial degradation. Are the microbes breaking up the oil? Can you detect an increase in microbial growth?

Oil degraded by microorganisms begins to appear darker and less slick and no longer covers the entire surface of the water. It is also less transparent and less oil appears on the surface. Microbial growth and oil breakdown can be detected by measuring the turbidity of the water.

Is the oil over the surface completely degraded? Can you still see any oil remaining on the surface? If so, explain.

The oil on the surface is not completely degraded and remaining oil is still visible on the surface. Four days did not allow the degradation process to complete and the smaller area may have caused the oil to be more concentrated than in an actual spill.

What happens to oil when it is biologically degraded in the ocean?

Oil that remains after evaporation goes through an emulsification process that creates a highly viscous, sticky material. This material becomes thick enough to sink to the bottom of the ocean. It sticks to anything it comes in counter with including marine wildlife. Oil left after emulsification is broken down by microorganisms and photo-oxidation. After 3 months only 15% of the original oil volume remains and it forms tarry clots that float to shore.

What is the purpose of the nutrient fertilizer used over the oil spill?

Nutrient fertilizer is used to enhance and speed the degradation of oil by increasing the bacterial and fungal growth.

Are there any adverse effects of using a fertilizer over an actual oil spill to enhance indigenous microbial growth?

Bioremediation is perhaps the most environmentally safe process of oil removal used today. It uses indigenous microbial growth so it does not introduce any new species that may damage the strict ecosystem. An increase in this one type of organism may cause a slight unbalance in this ecosystem but compared to other methods it has virtually no effect on the environment.

Did you observe an increase in turbidity over time? Which of the two simulations is more turbid? Explain.

An increase in turbidity over time was observed. The second simulation was the more turbid because the fertilizer increased microbial growth and enhanced oil degradation.

Did you observe more fungal and bacterial growth in the test tubes or in the jars? Explain.

The jars showed more fungal and bacterial growth because of the nutrient fertilizer and there was more space for growth in the jar.

Bases on the information provided, do you think that the microorganisms would be affected by water temperatures? Would they follow the floating oil or be dissipated by shifting winds or currents? And if they did eat the oil, would the residue damage marine life?

The microorganisms would definitely be affected by changes in temperature. These organisms grow best at fairly warm temperatures, and growth would be minimal in colder conditions. The microorganisms grow directly on the oil and would therefore not be dissipated by shifting. No harmful residue has been found after using bioremediation. All that is left after this method is masses of food and non-toxic living cells.

Based on the physical characteristics of oil and water discuss possible resulting problems associated with oil spills.

Because oil and water do not mix, oil can be spread over vast areas of oceanic surface if it is not contained. After emulsification, oil forms a thick, viscous substance that adheres to anything it comes in contact with. Also oil prevents light from reaching the lower areas of the ocean and reduces plant growth.

In this investigation, we evaluated the ability of microbes to degrade oil under optimum conditions. Based on your findings, discuss possible environmental limitations in using such a method over an actual oil spill in the ocean.

The temperature at an actual oil spill site could not be controlled and may drop below the microorganisms’ range of temperature that it can live. Also lower temperatures may reduce the rate of degradation. The oil may be further spread and dispersed by shifting winds making it harder for the microorganisms to cover its surface.

If you had to decide which clean up method to use in an actual spill, would you use such a bioremediation method or use a mechanical method described in the introduction? Explain your decision.

In an actual oil spill situation, a combination of bioremediation and mechanical methods would probably be most effective. Absorbent materials such as cloth, straw, powdered clay, or pine bark, could be used to contain the oil spill, while bioremediation is allowed to break down the oil into its harmless components.

Assuming that you need 10-6 lbs. of highly concentrated cell mass mixed with the nutrient fertilizer for the degradation of oil covering 0.022 sq. ft, as in the simulated oil spill, estimate the amount of cell mass and fertilizer mixture in pounds that would be needed to degrade the oil covering 1 square mile of an ocean. Assume there is the same amount of oil relative to the area.

12,672 lbs. would be needed to degrade one square mile of ocean.

Investigation #3: Simulated Oil Spill along the Shore

Describe the physical and chemical changes of the oil after several days of microbial degradation.

The oil began to get darker and lose its slick qualities. It no longer covered the entire surface of the water and was separated into sections. The chemical changes that occur, are that the carbon bonds are oxidized and broken into smaller hydrocarbon chains.

How effective do you think such a method is when used in an actual oil spill on the shore?

The dryer, sandier areas would slow the degradation process and inhibit the microorganisms from reaching parts of the oil spill, however, although slower, the method would still be fairly effective.

Discuss the physical limitations of using a mechanical method to clean up an oil spill on the shore. What are the limitations of using a bioremediation method? Which of the two methods would be most efficient and economical to clean up oil spills?

Mechanical methods would be virtually impossible to use in shoreline cleanup especially without further damaging the local wildlife. Rocks and other physical barriers would prevent skimming or absorbing the oil. Bioremediation would also be slightly hindered by these physical barriers, but it would still be the most efficient and economical way to cleanup a shoreline area.

Discuss the effect an oil spill on the shore would have on plant life along shorelines, protists and other larger animals.

Oil components are usually toxic to most organisms. Marine birds and mammals are especially effected by oil spills. They lose their ability to float on water and their insulation against cold, which can leave them severely handicapped or dead.

In the Alaskan oil spill, chemical detergents were not used. Why? Explain the use of detergents.

Chemical detergents were not used in the Alaskan oil spill because most of these chemicals are toxic to benthic, littoral, and open water marine life.

Discuss the potential of bioremediation procedures in detoxifying the air, water, soil, and waste materials.

Bioremediation has great potential for cleaning oil out of any type of medium as long as the microorganisms are able to grow in that environment. The microorganisms may be able to be genetically modified to grow on other mediums and even become airborne, however genetic engineering is still very controversial at this time.

What can be done to prevent oil spills.

Oil spills could be prevented by strict government regulation of all oil transportation and general public concern for the subject. Carefulness is the main key in preventing oil spills since most are caused by human error. Other methods of prevention could include new fuel sources or other means of transportation such as aircraft or truck.

How are we affected by oil spills? Discuss the physical, environmental, and economic consequences of oil spills.

Oil spills cause severe damage to the environment which can in turn effect humans. Oil spills are extremely wasteful and can cost millions of dollars to clean. Oil is also a non-renewable resource, which means that every time an oil spill occurs tons of oil are lost forever.

Research other energy sources that can be used to cut our dependence on oil?

Hydroelectricity, solar power, and wind power are all alternative energy sources that could reduce our dependence on oil. However, none of these sources are efficient enough to replace our need for oil. Nuclear power plants have began to grow, but these plants are highly expensive and dangerous. A last alternative to oil, could be gasohol, which is an alcohol made from corn that can be burned for fuel.

Error Analysis

Errors in this lab could be caused by incorrect measurements, insufficient oxygenating of the specimens, or mishandling of the sterile equipment. The tubes in Investigation #1 were not consistently inverted. The potency of the Penicillium culture was also questionable and there was no visible fungal growth in the original Luria broth culture.

Discussion and Conclusion

Investigation #1 acted as a control for the next two simulations. It showed that microbial growth and degradation did occur in a controlled environment. It could be used as a comparison to the amount of oil degraded in the other simulations to see if the variables had any effect on oil degradation. Investigation #2 was a simulation of an actual ocean oil spill with the use of a nutrient fertilizer. The nutrient fertilizer did speed oil degradation and a large amount of the oil was completely degraded in both jars. In Investigation #3, a shoreline cleanup was simulated with the addition of sand to the petri dish. The sand slightly hindered the effectiveness of the microbial degradation, but it was still effective in the partial breaking down of the oil.

All three investigations showed that the Pseudomonas culture was slightly more effective than the Penicillium culture in oil degradation. This may have been caused by a higher rate of reproduction in bacteria than in fungi.

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secrets of straight-a students

Set priorities. There is no interruption of study time with TV, phone calls, snacks, etc.
Study anywhere or everywhere. Keep a regular time everyday for studying and utilize extra time at work or play to study.
Get organized. Keep an organized planner and notebook with all assignments and an organized study area with all supplies. Make sure to have all assignments and needed materials with you each time you go to class.
Learn how to read. The secret of good reading is to be an “active reader.”
Schedule your time. Procrastination is a student’s best enemy. Don’t wait to the last minute to complete assignments or to study.
Take good notes – And use them! Always read the chapter, but listen and copy notes in class and then reread and recopy your notes each day.
Clean up your act. Neat assignments are more likely to get higher grades than sloppy ones.
Speak up. Participate in class by asking questions and showing intellectual curiosity.
Study together.
Test yourself. Make up and answer possible test questions or write out answers to chapter objectives.
Do more than you are asked. Part of learning is practicing and the more you practice, the more you learn.

The most important ‘secret’ of the super-achievers is not so secret. For almost all straight-A students, the contribution of their parents is crucial …Parents impress the lesson of responsibility on their kids.

Sample 6a Transformation Lab

Lab 6A – Bacterial Transformation & Ampicillin Resistance

Introduction:
Bacterial transformation occurs when a bacterial cell takes up foreign DNA and incorporates it into its own DNA. This transformation usually occurs within plasmids, which are small circular DNA molecules separate from its chromosome. There can be 10 to 200 copies of the same plasmid within a cell. These plasmids may replicate when the chromosome does, or they may replicate independently. Each plasmid contains from 1,000 to 200,000 base pairs. Certain plasmids, called R plasmids, carry the gene for resistance to antibiotics such as ampicillin, which is used in this lab.

Plasmids function in transformation in two different ways. They can transfer genes that occur naturally within them, or they can act as vectors for introducing foreign DNA. Restriction enzymes can be used to cut foreign DNA and insert it into the plasmid vectors. The bacteria used in this lab were Escherichia coli (E. coli). It was ideal for this transformation study because it can be easily grown in Luria broth or on agar, and it has a relatively small genome of about five million base pairs.

Transformation is not the only method of DNA transfer within bacteria. Conjugation is a DNA transfer that occurs between two bacterial cells. A bridge is formed between the two cells and genetic information is traded. In transduction, a virus is used to transfer foreign DNA into a bacterial cell.

Hypothesis:
The transformed E. coli with the ampicillin resistance gene will be able to grow in the ampicillin plates, but the non-transformed E. coli will not.

Materials:
The materials needed for this lab were 2 sterile test tubes, 500 μL of ice cold 0.05M CaCl2, E. coli bacteria cultures, a sterile inoculating loop, a sterile micropipette, 10 μL of pAMP solution, a timer, ice, a water bath, 500 μL of Luria broth, a spreading rod, 4 plates: 2 ampicillin+ and 2 ampicillin – , and an incubator.

Methods:
One sterile tube was labeled “+” and the other “-“. A sterile micropipette was used to transfer 250 μL of ice cold 0.05M CaCl2 to each tube. A large colony of E. coli was transferred with an inoculating loop to each tube. The suspension was then mixed by repeatedly drawing and emptying a sterile micropipette. 10μL of pAMP solution was added to the cell suspension in the tube marked “+” and mixed by tapping the tube. Both tubes were immediately put on ice for 15 minutes and then soaked in a 42° C water bath for 90 seconds. The tubes were then returned to ice for another 2 minutes.

After the heat shock, 250 μL of Luria broth were added to each tube. The tubes were mixed by tapping. Two plates of ampicillin + agar were labeled LB/AMP+ and LB/AMP-. The two plates of ampicillin- agar were labeled LB+ and LB-. 100 μL of the cell suspension in the “+” tube were placed on the LB+ and the LB/AMP+ plates. 100μL of the cell suspension in the “-” tube were added to the LB- and the LB/AMP- plates. These were spread with a spreading rod that was sterilized by passing it over a flame after each use. The plates were allowed to sit for several minutes and then incubated over night inverted at 37° C.

Results:

LB+

(Positive Control)

LB-

(Positive Control)

LB/AMP+ (Experimental)

LB/AMP- (Experimental)

Bacterial Growth

lawn

3 colonies

No growth

Questions:
1. Compare and contrast the number of colonies on each of the following pairs of plates. What does each pair of results tell you about the experiment?
LB+ and LB- Both of these plates had a lawn of bacteria. This proves that the bacteria are capable of growing on the agar and that there was nothing preventing growth beside the ampicillin.

LB/AMP- and LB/AMP+ The LB/AMP- had no growth, but the LB/AMP+ had small growth. This shows that the bacteria was transformed and developed a resistance to ampicillin.

LB/AMP+ and LB+ The LB/AMP+ had less growth than the LB+. This shows that the transformation was not completely effective and only transformed some of the most competent bacterial cells.

2. Total mass of pAMP used = 0.05 μg

Total volume of cell suspension = 510 μL

Fraction of cell suspension spread on the plates = 0.196

Mass of pAMP in cell suspension = 0.0098

Number of colonies per μg of plasmid = 0.0294

3. What factors might influence the transformation efficiency? Explain the effect of each you mention.
Transformation efficiency could be affected by the size of the colony added to the solution. In a larger colony the efficiency would increase because there would be more receptive cells. Another factor would b the amount of pAMP added. The more pAMP added, the higher the efficiency. The amount of Luria broth added could also affect efficiency. If the amount of Luria broth was increased, the efficiency would decrease.

Error Analysis:
This lab had several steps, each giving the potential for error. All of the measurements had to be precise and accurate, and the heat shock timing was also a very complicated procedure. Error in this lab could have been caused by the concentration of the CaCl2 due to the fact that most of it was frozen.

Discussion and Conclusion:
The bacteria treated with the pAMP solution developed a resistance to ampicillin and were able to grow on the ampicillin+ plate. Those that were not treated with the pAMP were not able to grow on this medium. The plates with no ampicillin served as a control to show how the bacteria would look in normal conditions. Transformation is never fully effective, Only cells that are competent enough are able to take up the foreign DNA. Therefore, the ampicillin + plates showed less growth than the control plate.

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Seed Germination & Detergents

Detergent & Seed Germination

Introduction:

Seeds come in different sizes, shapes, and colors. Some are edible and some are not. Some seeds germinate readily while others need specific conditions to be met before they will germinate. Within every seed lives a tiny plant or embryo.The outer covering of a seed is called the seed coat. Seed coasts help protect the embryo from injury and also from drying out. Seed coats can be quite thin and soft as in beans or very thick and hard as in locust or coconut seeds. Endosperm, which is a temporary food supply, is packed around the embryo in the form of special leaves called cotyledons or seed leaves. These generally are the first parts visible when the seed germinates. Plants are classified based upon the number of seed leaves (cotyledons) in the seed. Plants such as grasses and grass relatives can be monocots, containing one cotyledon. Dicots are plants that have two cotyledons.

Seeds remain dormant or inactive until conditions are right for germination. All seeds need water, oxygen, and proper temperature in order to germinate. Some seeds require proper light also. Some germinate better in full light while other require darkness to germinate.When a seed is exposed to the proper conditions, water and oxygen are taken in through the seed coat. The embryo’s cells start to enlarge and the seed coat breaks open and root or radicle emerges first, followed by the shoot or plumule which contains the leaves and stem.

Many factors contribute to poor germination. Over-watering results in a lack of proper oxygen levels. Planting seeds to too deep results in the seed using up all of its stored energy before reaching the soil surface, and dry conditions result in the lack of sufficient moisture to start and sustain the germination process.

Objective:

The students will be able to describe how some environmental factors affect seed germination.

Materials:

Masking tape, Scissors, 3 ziplock bags, Marker, Forceps, Paper Towels, Metric Ruler, 3 colored pencils, 25 seeds, distilled water, 50 ml graduated, 1% detergent solution, 10% detergent solution, graph paper

Procedure:

Label the 3 zip lock bags: Control, 1% Solution and, 10% Solution.
Cut 6 square pieces of paper toweling to fit each bag.
Place 2 squares in each bag.
Distribute 6 seeds on each side of the paper towel between the plastic and towel.
In the control bag add 25 ml of distilled water completely moistening the paper towel.
In the 1% solution bag add 25 ml of 1% detergent solution making sure to completely moisten the towel.
Do the same to the 10% solution bag by adding 25 ml of 10% detergent solution.
Make sure all bags are sealed tightly.
Place the bags in a dark warm place designated by the instructor.
Write a hypothesis predicting the results of the experiment.
Examine the bags daily for 5 days. Record any changes that might have occurred. If the roots is visible the seed is considered germinated.
Record your date in the table below.
Do not allow your towels to dry out. Moisten each bag with the appropriate solutions in equal amounts.
Measure the root growth of each seed daily from the time it appeared.
Graph the data from the table using the colored pencils to represent each of the zip lock bags.

Number of Seeds Germinated

Day	Control	1% Detergent Solution	10% Detergent Solution
1
2
3
4
5

Average Growth of Germinating Seeds(mm)

Day	Control	1% Detergent Solution	10% Detergent Solution
1
2
3
4
5

Graph Title: ________________________________________

Analysis:

1. How many of the seeds germinated after 5 days in distilled water? ________. In 1% solution? _______ in 10% solution? ________.

2. Was there a difference in the number of seeds germinated?

3. In which of the three bags did seeds germinate faster?

4. What was the purpose of the control?

5. Did the detergent strength have an effect on the seed’s germination? If so What was it?

6. Was your hypothesis correct? Why or why not?

7. If it was not, what will you do now?

Sample 6B DNA Lab AP

Lab 6B – DNA Fingerprinting

Introduction:
Restriction enzymes are endonucleases that actually cut the phosphodiester bonds on the sides of deoxyribonucleic acid. These endonucleases recognize specific DNA sequences in double-stranded DNA, which is usually a four to six base pair sequence of nucleotides. The endonucleases then digest the DNA at these sites. The resulting product is usually fragments of DNA of various lengths. Some restriction enzymes cut cleanly through the DNA double helix while some produce uneven or sticky ends. By using the same restriction enzyme to cut DNA from different organisms, the sticky ends produced will be complementary and the DNA from the two different sources can be recombined. In humans, no two individuals have the exact same restriction enzyme pattern in the DNA except for identical twins. In DNA, the antiparallel strands are difficult to deal with considering the restriction enzymes cut from opposite directions. This is the reason for the complementary ends. The restriction enzymes are named according to a system of nomenclature. The first letter represents the genus name of the organism. The next two letters come from the species name. If there is a fourth letter, it stands for the strain of the organism. Finally, if there are Roman numerals, it represents whether that particular enzyme was the first or second etc. isolated in that category.
In the electrophoresis chamber, there is placed an agar gel. This gel has wells in it for the samples of DNA to go into. The agarose gel is covered in a buffer so that the DNA is in a neutral pH solution. That way, the DNA moves in the direction its charge forces it. Since the phosphate groups on the skeleton of DNA are negatively charged, the whole molecule takes on the negative charge. So, when the DNA is placed inside the gel and the electricity turned on so that the poles are drawing the DNA toward the positive side, it will move through the gel and separate according to the size of the fragments.

Hypothesis:
By way of electrophoresis, the fragments of DNA of lambda can be separated by the traveling of the fragments through agar gel according to fragment size; DNA fingerprinting has occurred.

Materials:
The materials needed for this lab are the following: an electrophoresis chamber, an agarose gel, lambda DNA digested with endonucleases, tracking dye, micropipette and tips, running buffer, and an electrical supply.

Methods:
Prepare the agar gel for the electrophoresis by microwaving it for the suggested amount of time. When the gel has sufficiently hardened, place it in the chamber, pour the running buffer over the gel and add the DNA samples into the wells with a micropipette. Next, set the correct voltage and turn on the electricity. Allow this to run until the DNA is almost to the end of the gel, but do not let it run all the way out. Next, obtain the stain and a staining tray and let the gel set in the stain for a while. Next, put the gel into distilled water so that the stain can be taken out of the gel itself, leaving the DNA stained a royal blue. Look at and measure the gel over a light box, and put data into the data table.

Data:

Table 6.1

HindIII
Actual base pairing sequence	Measured Distance (mm)
23,130	12
9,614	18
6,557	22
4,361	28
2,322	41
2,027	43
570(may not be detected)
125(may not be detected)

Table 6.2

EcoRI
	Measured Distance (mm)	Interpolated base pairs sequence	Actual base pair sequence
Band 1	12	13,500	21,226
Band 2	14	11,000	5,148 or 5,973
Band 3	26	3,700	4,269
Band 4	28	3,150	3,530
Band 5	43	815	2,207
Band 6	47	580	1,904
Band 7	49	500	1,587
Band 8	58	220	1,375

Questions:
Discuss each of the following factors:

Voltage used. If a higher voltage had been used, the DNA would have moved faster through the agar gel, and slower if the voltage was low.

Running time. If allowed to run longer, the DNA would have eventually ended up into the running buffer, and lost to the experiment. If not allowed to run long enough, the bands could merge and be unclear for reading.

Amount of DNA. If more DNA had been used, the bands would have been darker because more of the fragments would have traveled the same distance in the gel. The bands would only have been more distinct and distinguishable.

Reversal of polarity. Had the polarity been reversed, the DNA would have been drawn the other way through the gel, and ended up in the running buffer.

Two small restriction fragments of nearly the same base-pair size appear as a single band, even when the sample is run to the very end of the gel. What could be dome to resolve the fragments? Why would it work? I would take the endonucleases needed to get the two fragment sizes and run an electrophoresis experiment just using those two sizes. It would probably work because these two fragments just by themselves can’t or shouldn’t stay together all the way to the end of the gel.

What is a plasmid? How are plasmids used in genetic engineering? Plasmids are small rings of DNA. They are used in genetic engineering because it is considerably easier to manipulate them into taking up preferred genes than it is to change the DNA sequence of the whole cell.

What are restriction enzymes? How do they work? What are recognition sites? These enzymes are endonucleases that cut the phosphodiether bonds of the DNA. They only cut at specific proteins, the recognition site.

What is the source of restriction enzymes? What is their function in nature? They occur naturally in prokaryotes and are used to cut up invading viral DNA that happens to get through the cell wall and plasma membrane of the bacteria.

Describe the function of electricity and the agarose gel in electrophoresis. The electricity is used to pull the DNA in a certain direction so that it will separate. The gel is helpful because it is like a freeze frame that allows the fingerprinting to be visualized. This could not be done in liquid or any solid.

If a restriction enzyme digest resulted in DNA fragments of the following sizes: 4000, 2500, 2000, and 400 base pairs, sketch the resulting separation by electrophoresis. Show starting point, positive and negative electrodes, and the resulting bonds.

What are the functions of the loading dye in electrophoresis? How can DNA be prepared for visualization? The dye allows the DNA to be more distinct so that accurate measurements can be made in determining the distance traveled and the amount of bands.

Use the graph prepared from the lab data to predict how far (in mm) a fragment of 8000 base pairs would migrate. A piece of DNA of that size would probably run about 17.5 millimeters.

How can a mutation that alters a recognition site be detected by gel electrophoresis? If you ran the normal and the mutant at the same time, you could see the change in the band that would be in a different place because it wouldn’t allow the DNA to be cut in that place.

Error Analysis:
There were not too many errors that could have occurred in this lab, but some of the few include the adding DNA to the agar gel. The person transferring had to have a steady hand and good eyes so that the gel wasn’t poked and the DNA made it into the chamber without problems. The wrong DNA samples were added to the wells, but the right ones were identified and later labeled correctly, out of order.

Conclusion:
In conclusion, DNA fingerprinting, or electrophoresis is used to determine the size of the fragments that are cut by restriction enzymes. Restriction enzymes only cut at their specific protein recognition sites. This is useful because no two restriction enzymes code for exactly the same recognition site, allowing for a “fingerprint” like uniqueness that is only possible with one’s DNA. From the data collected in the electrophoresis experiment, other sizes of parts can be hypothesized by following the size of the base pair to the line of best fit drawn on the log sheet. This tells you about how many millimeters the base pair would probably go if allowed the same circumstances.

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