Introduction: (Initial Observation)
Antibiotic is a substance, such as penicillin or streptomycin, produced by or derived from certain fungi, bacteria, and other organisms, that can destroy or inhibit the growth of other microorganisms.
The first antibiotic, penicillin, was discovered about seven decades ago. Sir Alexander Fleming discovers the drug penicillin, which counteracts harmful bacteria. Fleming makes the discovery by accidentally contaminating a bacteria culture with a “Penicillium notatum” mold. He notices that the non-toxic mold halts the bacteria’s growth, and later conducts experiments to show penicillin’s effectiveness in combating a wide spectrum of harmful bacteria.
In this project we will investigate the effect of antibiotics on bacteria count.
The procedures and experiments that I am proposing here are for actual bacteria count. If you just want to see the effect of antibiotics on bacteria growth, you can do it much easier.
Your test media can be a cup of chicken broth, mixed with some sugar. When the bacteria grow, they will create gases resulting a very bad odor. They also convert sugar to acid and drop the pH in a solution. So you can simply get some chicken broth (canned powder), dissolve it in water, add sugar and a few drops of polluted water. then you divide your sample in two parts. Add some antibiotic to one part. cover both with a filter paper or aluminum foil. Keep both samples in a warm place such as an incubator for 24 hours. Check both samples to see which one smells bad or has a lower pH. That is the one with a higher level of bacteria.
Information Gathering:
Find out about bacteria growth. Read books, magazines or ask professionals who might know in order to learn about the effect of antibiotics on bacteria. Keep track of where you got your information from.
Read me first:
Growing bacteria is one of the most rewarding and educational activities that a student can do. You will learn a lot of things just by growing bacteria. We usually grow bacteria as a way of identifying or counting bacteria. Bacteria can grow anywhere as long as food, moisture and proper temperature is available. Proper temperature for growing most bacteria is about 37 to 40 degrees centigrade. This is the same as body temperature of warm blooded animals. That does not mean that bacteria do not grow in other temperatures. If the temperature is not perfect, bacteria will grow slower.
If you have ever placed some cut flowers into a clear jar, you may have noticed that in a few days the water becomes cloudy and smells bad. Bacteria are the cause of cloudiness in water. Recently this level of cloudiness is measured by special machines and used as a method of counting or estimating the amount of bacteria (bacteria count). This method is not accurate, but it is fast and does not need a 24 hours or more waiting time.
To accurately count bacteria in a sample, first a dilution of the sample is made. Then the dilution is spread on a nutrient agar petri-dish for bacteria growth. Each bacteria will reproduce and become a bacteria colony. So we can simply count the colonies.
I said nutrient agar, because agar by itself is not a food for bacteria. You need to add some food to the agar to make it nutrient agar. What food is good for bacteria? Think a moment. What foods spoils faster and create the worst odor? They are most likely good for bacteria. I usually use some fat free chicken broth or beef broth as food. I may also add a small amount of sugar. If you want to do this, make sure you filter the broth so it will be clear. Chicken broth powder can be purchased from supermarkets, the only problem is that they also contain some flavor and vegetables so they can be served as soup. If you use them, you still need to filter them with a coffee filter.
But why do we use agar? We use agar because agar can form a gelatinous moist and clear medium for growing bacteria. There are a few reasons that you can not use gelatin itself. The first reason is that gelatin melts in warm temperature, so you have to keep it cold and bacteria don’t grow fast in cold temperature. The other reason is that I think manufacturers of gelatin add some preservatives that stop or slows down the bacteria growth. So far agar is the best known gelatinous substance for growing bacteria.
Since agar is also used as a food additive, you may purchase agar from health food stores or whole food stores. You may need to do some search on that. Some of these stores don’t know what the agar is.
So start today. Prepare your broth, add some agar, let it boil for a few minutes and fill up your petri-dishes or any other thing that you want to use for bacteria growth, Keep it open for a few minutes so the bacteria from the air will get to that. Cover it and keep it in a warm place for 24 hours. you should then be able to see the bacteria colonies.
A few notes that may help:
Agar concentration must be 1 to 3 percent to get a good gel. I think you better do it with 2% agar. Same amount chicken broth and half of that sugar must be sufficient. In each petri dish add enough agar to cover the bottom of the dish. Usually you cover the petri-dish and keep it upside down in a warm storage such as incubator. When petri dish is upside down, agar does not dry and condensation does not form on the petri-dish cap.
Have fun with your experiments.
If you are growing household bacteria, you can just dump you used petri-dishes in garbage and wash everything else with warm water and liquid detergent.
Visit Microbiology primer for lot’s of valuable information and samples.
Determining which antibiotic is most effective
This is how we determine in the laboratory which antibiotic will kill a particular organism like E. coli. The picture that you see below is showing a culture plate with bacteria and antibiotic disks. The bacteria was spread evenly all over the culture plate. Next, little white disks with different antibiotics in them were dropped on the plate. Each white disk represents a different antibiotic like ampicillin, tetracycline, gentamicin, and others. The culture plate was then placed in an incubator for 18-24 hours to allow the bacteria to grow. If the bacteria is sensitive to a particular antibiotic, it will not grow close to the disk. If the bacteria is resistant to an antibiotic, it will grow right up to the disk.
In the picture above, the bacteria is sensitive to all the antibiotics. Some organisms are quite resistant to antibiotics, thus, the need to perform sensitivity studies such as the one you see here. Without laboratory tests such as this, the physician would be guessing at which antibiotic to use. Only medical laboratory technologists are licensed to perform and interpret such tests.
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.
The purpose of this investigation is to see the effect of antibiotics on bacteria count.
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.
If you are going to do this experiment in an advanced biology lab with all possible equipment, your independent variable is the amount of antibiotic that you use. But for now the variable is antibiotic (presence, absence).
Dependent variable is the bacteria count.
Constant is the type of antibiotic.
Controlled variable are temperature, light and any other factor that may affect the bacteria growth in our different experiment runs.
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.
I think antibiotic can reduce the bacteria count to zero unless there are some antibiotic resistant bacteria in our test sample. I also think that the amount of antibiotic or the ratio of antibiotic to bacteria is important because if antibiotic is not enough to disable all bacteria, then bacteria may get chance to mutate and become resistant to 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.”
If an antibiotic be able to kill bacteria or disrupt the growth or reproduction of bacteria, we will have a lower bacteria count on samples exposed to antibiotic.
PROCEDURE:
For your experiment, you will get a sample containing bacteria and divide it in two parts. Expose one part to antibiotic and then test bacteria count in both parts. To count the bacteria, grow bacteria on nutrient agar plate. Each bacteria will grow to a colony in about 24 hours. That’s when it is visible and you can count them Each colony represents one bacteria in the test sample. Note that some times there are millions of bacteria in a very small sample. If so many new colonies grow on a petri-dish, we will not be able to count any thing. That is why we dilute our sample using distilled water. We make many different dilutions such as 1:1000 and 1:10,000 and 1:100,000 and 1:1000,000. Then we do bacteria count test on all of them, hopping that in one of them bacteria colonies will be in a countable quantity.
Step 1:
Prepare 24 culture media plates for growing bacteria. You may purchase a bacteria culture kit and prepare your plates using the agar that comes in the kit.
| Image on the write shows the kit contents from MiniScience.com. |  |
If you have access to the following material, make your own nutrient agar using the following formula and then use that to prepare your plates.
TGY Tryptone Glucose Yeast
- Tryptone…………. 5.0g
- Agar……………. 10.0g
- Yeast extract…….. 5.0g
- Glucose………….. 1.0g
- K2HPO4…………… 1.0g
- Spring water……1000 mL
This is called Plate Count Agar by many people. It supports more species of bacteria than any other medium. It is called TGY. Bacterial pigments which are pale on some media are usually much brighter on TGY in air at room temperature. Used as slants and stabs. You can also add 1 gram of powdered CaCO3 to counteract the acid generated by many bacteria from the glucose. With the carbonate, many stock cultures last years instead of months. If you don’t have calcium carbonate powder try ground agriculural limestone or any limestone or blackboard chalk. To avoid settling of CaCO3, stir while filling tubes. The low level of glucose helps reduce acid production and cultures live longer.
This is the only medium a teacher or student needs. You can do lots of interesting work using only this medium.
If you do not have petri dishes, nutrient agar or a kit for that purpose, click here to learn how you can substitute these material.
Step 2:
Get a sample of polluted water for test. Mix 2 ml of polluted water with 10ml chicken broth in a test tube and incubate it for 24 hours so the bacteria will reproduce and increase. Usually this is done on a device that constantly moves, so the bacteria can freely move in the liquid. Most likely you will not have a vibrator, so it is good if you shake the test tube a few times during this incubation period.
Step 3:
While the bacteria are being incubated, prepare some antibiotic disks as described here. (Antibiotic disks can also be purchased from biology suppliers).
Break an antibiotic capsule (I used Ampicilin) and empty the contents in a clean petri-dish. One capsule will be enough for hundreds of disks.
Dispose of the plastic shell and add a few drops of water to the remaining powder. Cut some filter papers in small pieces and soak them in the antibiotic solution. Let the disks dry in a clean space. You may cover them with another filter paper to protect them from dust.
Although they are known as antibiotic disks, you can cut them in small squares.
The reason that we use filter paper, is that other papers often have starch and other polymers that may affect the results of our experiments. Filter paper is pure cellulose fiber.
Step 4:
Use the bacteria that you grown in step 2 and prepare different dilutions of bacteria.
- Prepare 1:10 dilution of the sample. To do this, take 2 mL of the sample and blend it with 18 mL of distilled water.
- Prepare a 1:100 dilution of the sample by taking 1 mL from previous solution and adding 9 mL of water to it in a test tube.
- Prepare 1:1000, 1:10,000 and 1:100,000 dilutions by taking a 1 mL of solution from previous dilution and adding 9 mL of distilled water
- Pipette 0.1ml of each dilution onto a Plates Count Agar (PCA) plate
- Take a glass hockey stick submersed in ethanol and run it through a flame to sterilize it. (Glass hockey stick is a glass rod bent on one end like a hockey stick. It is used to spread bacteria on the surface of agar plate. You may use a steel spoon instead.)
- Let it cool and use it to spread dilution around the plate
- Do this on two plates for each of the five different dilutions.
- Place an antibiotic disk on one of the plates of each dilution. Label the plates with dir dilution level.
- incubate all plates at 35 degrees Celsius for 24 hours and then count the bacterial colonies.
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Record the results in a table like this:
| Dilution | Bacteria count for plate with antibiotic disk | Bacteria count for plate without antibiotic disk |
| 1:10 plates | 8 | 120 |
| 1:100 plates | 6 | 55 |
| 1:1000 plates | 6 | 71 |
| 1:10,000 plates | 7 | 97 |
| 1:100,000 plates | 8 | 112 |
How do you determine the actual bacteria count?
If a 0.1ml of 1:100 dilution shows 7 bacteria colony, then:
- There has been 7 bacteria in 0.1ml of 1:100 solution.
- There has been 700 bacteria in 0.1ml of the original solution (before diluting).
- There has been 7000 bacteria in 1ml of the original solution.
Analyze your results:
The above table contains a set of sample results in gray. This sample of results show that the bacteria counted are not from the samples. Instead they are from the environment or from the nutrient agar plates. Where the bacteria are from samples, they must match the dilution pattern. For example if you have 7 bacteria in your 1:1000 sample, you must have about 70 bacteria in your 1:100 sample and about 700 in your 1:10 sample. (You see that sanitation and clean environment is crucial to the bacteria growth tests.)
Disregarding the source of bacteria, the effect of antibiotic is clear. Take the average of bacteria count with antibiotic and without antibiotic and use them to make a graph.
Use a bar graph:
You can use a bar graph to visually present your results. Make two vertical bars. Label one bar “With Antibiotic”. Label the other bar “Without Antibiotic”. The height of each bar must show the average bacteria count on that group.
Materials and Equipment:
- 10 test-tubes of sterilized water
- 10 PCA plates
- blender or mixer (optional)
- Bunsen burner
- graduated cylinder
- Ethanol (Used for sterilizing. Just flame is enough in most cases)
- glass hockey stick
- pipettes
- refrigerator
- incubator (A warm cabinet for growing bacteria)
- microwave
- scale
- large beaker
- hot plate
- Sample anti-biotic
Note: List of material depends on your final procedure design. You may modify these procedures based on what is available to you.
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.
You may change the procedures suggested above in order to adapt it to your equipment, supplies or questions. For example here initial sample of polluted water was placed on a nutrient agar plate and incubated for 48 hours. (The nutrient was chicken broth with small amount of sugar.) The bacteria colonies appeared to be in two different colors. So I decided to grow the bacteria with a yellow colony. I used a sterile spatula to remove one colony and transfer it to 2 ml of distilled water in a test tube.
Then I took 3 nutrient agar plate and added 0.5 ml of the solution on each of the plates. I left one plate without any antibiotics, placed one antibiotic disk on the second plate and two antibiotic disk on the third plate. All plates were incubated for 48 hours. Images show that no bacteria is grown close to antibiotic disks.
What Nutrient Agar dir I use?
The nutrient agar in these plates was made using 9 grams of chicken broth (powder), 5 grams of Agar and 500 ml water. I also used a few drops of food coloring hopping that it will make the bacteria colonies more visible, but I did not feel any difference. That made me 15 nutrient agar plates that I kept in refrigerator for later use.
How to dispose the bacteria infected plates?
One way is placing all the plates in an autoclave in the temperature of 130ยบ for one hour. Note that this temperature is under pressure and hot steam will kill bacteria. Dry hot air does not kill the bacteria at this temperature.
Another way is using antibacterial disinfectants. Soak the plates in a strong antibacterial solution for a few days and then dispose them.
Calculations:
Depending on your method you may or may not need to do any calculations. If instead of bacteria disk you use bacteria solution, then you may also want to do some calculations to find out how many bacteria will be killed by the action of certain amount of antibiotics.
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.
Question:
rather than using just polluted water I was interested in using a bacteria sample from either Carolina.com or Sciencestuff.com… there were many to choose from and I could use some direction on what to select. I plan on using at least three to five different antibiotics and making my own disks.
I don’t know which antibiotics yet. I am going to visit our doctor and ask for some samples. I did some research and was going to ask for Penicillin, Cipro, Tetracycline & chloramphencol but will take what I can get. I will be getting my science teachers permission for the bacteria samples and having them shipped directly to school. are there some particular bacteria that you would recommend.
My choices are: (first list from sciencestuff.com) each is available individually.
Acetobacter Aceti
Acinetobacter Calcoaceticus
Alcaligenes Fecalis
Azotobacter Chroococcum
Bacillus Cereus
Bacillus Cereus – Var. Mycoides
Bacillus Megatherium
Bacillus Stearothermophilus
Bacillus Subtilus
Branhamella Catarrhalis
Citrobacter Freundii
Corynebacterium Pseudo-diptherium
Enterobacter Aerogenes
Escherichia Coli
Lactobacillus Acidophillus
Micrococcus Luteus (Yellow)
Micrococcus Roseus (Red)
Pseudomonas Fluorescens
Rhizobium Leguminosarum
Rhodospirillium Rubrum
Sarcina Aurantiaca
Sarcina Lutea
Sarcina Sublava
Serratia Marcescens (Red)
Serratia Marcescens – Strain D1
Serratia Marcescens – 933
Serratia Marcescens – WCF
Spirillum Volutans
Streptococcus Lactis
Penicillium Notatum
Vibrio Fisheri (Photobacterium)
Or as a set from Carolina.com
Bacillus megaterium
Branhamella catarrhalis
Micrococcus luteus
Pseudomonas fluorescens
Rhodospirillum rubrum
Serratia marcescens
Sporosarcina ureae
or is it possible to duplicate any of the above from common sources?
What would you suggest?
Answer:
Purchasing bacteria is not required for this project. Thousands of household bacteria are available in our skins, mouth, kitchen counter, foods, refrigerator door and bathrooms. More dangerous bacteria are available in the soil.
The only benefit that you get from purchasing bacteria is that they are usually isolated and known. Almost all those bacteria can also be found at home, but they are usually mixed with other bacteria. For example Lactobacillus Acidophilus can be found in a number of brands of yogurt and in the form of powders, capsules, tablets and liquids which are available in health food stores. I have no specific suggestion on selecting bacteria from the distributors list. You may want to study on those bacteria and select those who you can visually distinguish them based on color or shape of colony.
To find images of these bacteria you can search for them in Google.com and select Images.
