Introduction: (Initial Observation)
There are many causes of mutation and some of them can lead to complex and unusual types of mutations. Mutations in bacteria also happens naturally for the purpose of adaptation to new environmental factors. This type of mutation is called directed mutation. Also external factors such as radiation can potentially result mutations. The most common form of mutation is caused by antibiotics, resulting new strands of the bacteria that are resistant to the antibiotic.
The experiments in this project requires bacteria growth. You need to be familiar with bacteria growth in flasks and in petri-dishes with growth media. I will include some information in this page; however it is also good to look at general bacteria growth experiments in this website or other educational websites.
Information Gathering:
Find out about bacteria growth and cell mutation. Read books, magazines or ask professionals who might know in order to learn about the factors that may cause mutation. Keep track of where you got your information from. Following are samples of information that you may gather.
About antibiotics:
Up until the Second World War, bacteria had things very much their own way. Only the immune system stood between humans and thousands of disease causing bacteria. Then Howard Florey in England showed that penicillin, a compound produced by one of Sir Alexander Fleming’s molds, could be sent into battle against these tiny invaders.
Use of penicillin marked a major turning point in therapeutic medicine, and antibiotics have become our first line of defense against many diseases.
Unfortunately, natural selection and evolution have prevented antibiotics from wiping out illnesses caused by bacteria and giving us a disease-free world. Despite their simplicity and apparent genetic homogeneity, many bacteria carry small circles of DNA on which genes can change rapidly.
These genes can mutate and alter their function without harming the ability of the bacteria to grow and reproduce. In this way, they provide a pool of variation somewhat similar to the variants of genes found in higher organisms.
When an antibiotic floods a person’s body, millions and millions of sensitive bacteria die, taking their genes with them. Somewhere, however, in the vast population of bacteria in the human body, one cell has just the right mutation to change a protein in just the right way so it can fight off the killing action of the drug.
Once this protein in altered, the bacterium can live in the presence of the antibiotic. Resistance to the killing action of the chemical enables this one cell to grow, divide and reproduce under the new environmental conditions in which all its relatives have died.
The gene for antibiotic resistance, therefore, has a positive effect on the survival of a bacterium when the antibiotic is present.
Unharmed by the new environmental circumstances, the resistant bacteria take over and dominate the population by growing and dividing unchecked. Eventually, all the bacterial cells become resistant, rendering that particular antibiotic useless as a therapeutic agent for that person.
Some forms of syphilis, a worldwide killer, cannot now be controlled by penicillin any longer. All the cells causing this disease are now resistant. Medical authorities regard the steady increase in bacterial infections that cannot be treated with antibiotics as the most serious problem facing world health.
Although bacterial resistance to antibiotics sounds like a classic example of natural selection at work, the results could be explained in another way. The antibiotic itself could have acted on the bacterial population and caused a shift towards antibiotic resistance.
What is mutation and how does it happen?
Mutations (mew-tay-shuns) are misspellings or changes in the genetic code. They’re pretty rare; only about one mutation occurs for every 1-10 million DNA bases.
But microbes reproduce very quickly. For example some bacteria can divide every nine minutes. And the entire DNA string or genome (gee-nome) of microbes is relatively small. For example, the average bacterium has only about five million DNA bases. So in each new generation of bacteria, there may be one or two mistakes in the genetic code.
In other words, these random mutations occur often enough in microbes to play a significant role in their ability to survive and adapt.
Some mutations are random “mistakes.” They can occur when a microbe is exposed to radiation or chemicals that cause changes in DNA. Microbes, like us, have DNA repair proteins that, like a mini construction crew, work hard to repair any such mistakes. They don’t always catch every mistake, however.
Other mutations result from a change in a creature’s circumstances. For example, if the temperature starts getting hotter and hotter, “jumping genes”—pieces of DNA that can move around—may pick up and move to a new place in the microbe’s DNA. This switch may result in giving the microbe a new genetic trait—like greater heat tolerance—that helps it adapt and survive better. It’s like a homebuilder who, upon realizing that he’s building in a hurricane-prone area, decides that the heavy oak wood he planned to use for hardwood floors is more urgently needed to build the beams that support the house, so he changes it on the blueprint. Jumping genes cause genetic changes in all kinds of microbes.
Another way bacteria can undergo mutation is to take up bits of free-floating DNA in the environment. This is called transformation. Bits of DNA litter the environment when it’s released from dying bacterial cells or bacteria burst open by a virus infection. DNA in the form of plasmids—circular bits of DNA that contain genes but exist outside the main chromosome—are also released by bacteria into the environment where they can be taken up by other bacteria.
Sometimes mutations are harmful and the microbes die off before they can reproduce and pass the mutations on to offspring. But other times a mutation gives rise to a new trait that helps the microbe better survive. Take antibiotic resistance among bacteria.
Antibiotics (an-tea-bye-ah-ticks) are life-saving drugs that kill bacteria. We discovered and began using them about 60 years ago. In that relatively short amount of time, bacteria have developed new traits through mutations that help protect them against antibiotics—sort of like a helmet protects you against injury.
Other mutations have helped microbes adapt to all sorts of environments from salty to icy to extremely hot and live off everything from decaying leaves to sunlight to bubbling sulfur.
Mutation rate in bacteria….
- Antibiotic-resistant bacteria become selected for in nature very rapidly. This is partly because bacteria are very small and thus small volumes hold huge numbers of bacteria.
For example:-
The “normal” mutation rate for a gene in nature is one mutation in every million to every billion divisions. - One mL of bacteria contains approximately 1,000,000,000 bacteria.
- So in one mL there are 10 bacteria with a mutation in that one gene.
- But, a typical bacterium has 3500 genes, which means that in each mL of bacteria there are approximately 35,000 mutations!
Thus, exposing bacterial populations to antibiotics is a perfect route to select resistant mutants!
Abstract: (Sample)
It has been proven by scientist that after being treated by certain antibiotics, bacteria often mutates and becomes immune to the antibiotic. These mutations are of particular interest to the scientist, because of the harm they would do to the community. For, if the bacteria were to mutate and become immune to the only antibiotic available, soon there would be nothing to stop it. This project’s objective it to see if this is really true and also to calculate that mutation rate. This objective was achieved by a number of activities and experiments. Mostly, though, the hey was observation. Some bacteria was grown and different solutions of streptomycin were added, then formulas were used to do the calculations. All these procedures did not take long, but waiting for the bacteria to grow was time-consuming. In the end, a mutation rate was settled upon and it was discovered that the rate was very small- approximately 7.2218*10 -9. It is not very significant now, but it may increase over the years. It was found that bacteria sometimes does mutate in bacteria, and that one day we will be out of antibiotics that work on specific bacteria.
About bacteria growth experiments
The following process can be used to determine if there is any live bacteria in a solution. It can also be used to isolate and grow a single type of bacteria.
Get a sample of water from a pound or stream. It is clear. Does it contain any bacteria? Bacteria can not be seen by naked eye. If they are not many, you cant even use a microscope to see them. So how do you know if there is any live bacteria in your sample water?
Although bacteria can not be seen, a colony of bacteria can easily be seen. You just need to grow the bacteria on nutrient agar plates. Nutrient agar plate is a petri dish that its bottom is covered by a gel made of agar and some nutrients for bacteria growth.
Initially your nutrient agar plate looks like this. It can be clear or it may have some color depending on the nutrients mixed with the agar. For simple experiments I use no fat chicken broth, water and agar. Such agar plate is clear with pale yellow tint.
Place one drop of bacteria infected water (or any other liquid) on the plate and use a sterile object to spread it. Initially wet liquid is noticeable because of its light reflection.
After a few minutes water dries and you see nothing on the petri dish. There might be a few live bacteria on the plate, but they are so small that can not be seen. Place your petri-dish (nutrient agar plate) in an incubator and look at that after 24 hours or 48 hours.
After being in incubator for one to 3 days, each bacterium splits and form a colony of thousands of same type bacteria. Bacteria colonies are viewable by naked eye. The shape- size and color of each bacteria colony varies based on the type of bacteria; however each colony contains only one type of bacteria.
If you leave the plates in the incubator for a longer time and if there are enough nutrients in your agar, bacteria colonies will grow larger.
To have a pure sample of any of the bacteria, you can use a sterile tool to remove some bacteria from any specific colony and transfer it to sterile water or any nutrient solution.
If you take a sample of this latest solution and grow it on another agar plate, you will notice that all bacteria colonies are the same shape, color and almost the same size. This shows that all bacteria in the solution are the same type.
You can then use this solution of a single type bacteria for your other experiments such as mutation experiment.
Note: You grow bacteria on agar plate only if you want to observe or separate certain colonies. Otherwise you grow bacteria in flasks or test tubes containing nutrients. No agar will be required.
Question/ Purpose:
What do you want to find out? Write a statement that describes what you want to do. Use your observations and questions to write the statement. Following are two questions proposed for this project.
Question 1: Does antibiotic cause mutation or antibiotic resistant mutants already exist in a bacteria colony? For this question you will see the experiment 1 and its results. You may optionally try to repeat the experiment and compare your results with the reported results.
Question 2: Can low dosage of antibiotic contribute to the production of antibiotic resistant mutants? This is the subject that you may study on your own. Suggested experiment is the experiment number 2.
Identify Variables:
When you think you know what variables may be involved, think about ways to change one at a time. If you change more than one at a time, you will not know what variable is causing your observation. Sometimes variables are linked and work together to cause something. At first, try to choose variables that you think act independently of each other.
Variables for the question number 2 are:
- The independent variable is the concentration of antibiotic used to destroy bacteria.
- The dependent variable is the rate of mutation.
- Controlled variables are (all other environmental factors that may affect growth, reproduction and mutation of bacteria) culture media, temperature and antibiotic.
Hypothesis:
Based on your gathered information, make an educated guess about what types of things affect the system you are working with. Identifying variables is necessary before you can make a hypothesis.
When the amount of antibiotic is not enough to destroy all bacteria, this gives chance to some bacteria to mutate and adapt to the new environment (presence of antibiotic).
Experiment Design:
Design an experiment to test each hypothesis. Make a step-by-step list of what you will do to answer each question. This list is called an experimental procedure. For an experiment to give answers you can trust, it must have a “control.” A control is an additional experimental trial or run. It is a separate experiment, done exactly like the others. The only difference is that no experimental variables are changed. A control is a neutral “reference point” for comparison that allows you to see what changing a variable does by comparing it to not changing anything. Dependable controls are sometimes very hard to develop. They can be the hardest part of a project. Without a control you cannot be sure that changing the variable causes your observations. A series of experiments that includes a control is called a “controlled experiment.”
Experiment 1: (For question number 1)
When an antibiotic resistant bacteria is discovered, there are two possibilities. One is that one or more bacterium have already had natural resistance to antibiotic, so they survived and reproduced. The other possibility is that the antibiotic itself could have acted on the bacterial population and caused a shift towards antibiotic resistance.
To test this alternative hypothesis, scientists carried out two experiments. A flask of growing bacteria was diluted with sterile salt solution and small samples were transferred to test tubes.
If the dilution was correct, each new test tube contained a single bacterial cell capable of growth into a new population. Before the bacteria began to grow, however, an antibiotic was added to each tube and all tubes were placed in a warm incubator under perfect growth conditions.
When the tubes were examined the next day, most of the bacteria had died, killed by the antibiotic against which they had no protection. But, every once in a while, one tube was seen to be filled with growing cells.
When these cells were tested, they all showed resistance to the antibiotic. So far, this experiment had simply duplicated the conditions seen in the human body and confirmed those results.
- Natural Selection 1. A single bacterium is placed in every test tube along with an antibiotic. Those bacteria with antibiotic-resistance genes survive, the sensitive bacteria die.
A second experiment, however, showed that variation followed by natural selection could explain these observations.
Starting again from the beginning, scientists placed single bacterial cells into separate test tubes. This time, however, they allowed the bacteria to grow into new populations before adding anything to the tubes.
Once each tube was full of bacteria (that had grown from a single cell), and antibiotic was added and all the sensitive cells were killed.
Occasionally, however, in one tube the antibiotic had no effect and all the cells in that tube were found to be resistant to the killing action of the drug, just as they had been in the first experiment.
Clearly, the second experiment shows that the gene for antibiotic resistance came first. A small variation in the base sequence of the DNA produced a variant gene that enabled the bacterium to defeat the killing action of the antibiotic. Alone in its test tube, this bacterium showed the “bottleneck” effect and passed on its genotype to all it’s descendants.
Later, when the antibiotic was added to the tubes, the cells without this saving gene died (they were less fit), but the descendants of the resistant cell lived on (they were more fit). Variation in the available genotypes followed by an environmental change (adding the antibiotic) had brought about the natural selection of the fittest gene combination
- Natural Selection 2. In this second experiment, each individual bacterium grows into a population of identical individuals before the antibiotic is added.
- As with the first experiment, the sensitive bacteria die and those bacteria with the mutated, antibiotic-resistance gene survive.
- This experiment shows that the mutation came first and was then followed by environmental selection.
Experiment 2: (For question number 2)
Introduction:
In this experiment you will expose identical samples of bacteria solutions to different amounts of anti biotic and test to see if you can find any antibiotic resistant mutant. Mutants must be able to grow in presence of antibiotics.
Procedure:
Prepare a solution of a single bacteria for this experiment. If you don’t know how to do this, read the instructions provided in the gathering information section.
Get 10 test tubes and add 2 ml of bacteria solution to each test tube. Number the tubes from 1 to 10.
Make solution of a common antibiotic such as Amoxicillin or Ampicillin by adding 500 mg of the antibiotic powder to 500 ml of water.
Add one drop of antibiotic solution to the test tube number 1.
Add two drops of antibiotic solution to the test tube number 2.
Continue with the same order and finally add 10 drops of antibiotic to the test tube number 10.
Allow the tubes remain in incubator for 24 hours.
Test one drop of the solution in each test tube for presence of live bacteria. (If you don’t know how to do this, read the instructions in the gathering information section)
Analyze the results:
If all 10 test tubes have live bacteria after 24 hours, either they are all resistant to the specific antibiotic that you used or the amount of anti-biotic that you have used has not been enough to kill all bacteria. To make sure you will need to repeat your test with higher amounts of anti-biotic or a different antibiotic.
If none of the test tubes have live bacteria after 24 hours, there has been no resistant mutants and no mutation has happened. In this case repeat your experiments with more diluted anti biotic solution.
Materials and Equipment:
List of material can be extracted from the experiment section. For antibiotics you may use any common antibiotic such as Amoxicillin or Ampicillin.
Results of Experiment (Observation):
Experiments are often done in series. A series of experiments can be done by changing one variable a different amount each time. A series of experiments is made up of separate experimental “runs.” During each run you make a measurement of how much the variable affected the system under study. For each run, a different amount of change in the variable is used. This produces a different amount of response in the system. You measure this response, or record data, in a table for this purpose. This is considered “raw data” since it has not been processed or interpreted yet. When raw data gets processed mathematically, for example, it becomes results.
Calculations:
No calculation is required for this experiment.
Summary of Results:
Summarize what happened. This can be in the form of a table of processed numerical data, or graphs. It could also be a written statement of what occurred during experiments.
It is from calculations using recorded data that tables and graphs are made. Studying tables and graphs, we can see trends that tell us how different variables cause our observations. Based on these trends, we can draw conclusions about the system under study. These conclusions help us confirm or deny our original hypothesis. Often, mathematical equations can be made from graphs. These equations allow us to predict how a change will affect the system without the need to do additional experiments. Advanced levels of experimental science rely heavily on graphical and mathematical analysis of data. At this level, science becomes even more interesting and powerful.
Conclusion:
Using the trends in your experimental data and your experimental observations, try to answer your original questions. Is your hypothesis correct? Now is the time to pull together what happened, and assess the experiments you did.
Related Questions & Answers:
What you have learned may allow you to answer other questions. Many questions are related. Several new questions may have occurred to you while doing experiments. You may now be able to understand or verify things that you discovered when gathering information for the project. Questions lead to more questions, which lead to additional hypothesis that need to be tested.
Possible Errors:
If you did not observe anything different than what happened with your control, the variable you changed may not affect the system you are investigating. If you did not observe a consistent, reproducible trend in your series of experimental runs there may be experimental errors affecting your results. The first thing to check is how you are making your measurements. Is the measurement method questionable or unreliable? Maybe you are reading a scale incorrectly, or maybe the measuring instrument is working erratically.
If you determine that experimental errors are influencing your results, carefully rethink the design of your experiments. Review each step of the procedure to find sources of potential errors. If possible, have a scientist review the procedure with you. Sometimes the designer of an experiment can miss the obvious.
