Introduction: (Initial Observation)
Ultrasonic are vibrations of frequencies greater than the upper limit of the audible range for humans that is, greater than about 20 kilohertz. The term sonic is applied to ultrasound waves of very high amplitudes. Ultrasound waves are vibrations that start from an ultrasound transducer and travel through air and objects by moving from one molecule to the next. Such vibrations in a high amplitude may potentially be able to kill bacteria or disrupt the process of bacteria growth or reproduction.
In this project we will investigate the effect of ultrasonic 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 ultrasonic 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. cover both with a filter paper or aluminum foil. Expose one to ultrasonic for one hour or more. 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 what you want to investigate. Read books, magazines or ask professionals who might know in order to learn about the effect or area of study. Keep track of where you got your information from.
Start by finding information about “what is ultrasonic?”. How is it made? and what are it’s properties and applications.
Read the report of U. S. Food and Drug Administration about Microbial Inactivation using ultrasound.
Read another report about different types of Ultrasound and their applications.
Following are some related information:
Ultrasound is a potentially attractive technique for the disinfection of water from bacteria. The major effects of ultrasound are due to cavitation, i.e. the rapid growth and explosive collapse of microscopic bubbles as the alternate compression and rarefaction of the sound waves pass through the liquid.
The attractiveness of ultrasound is enhanced by the fact that some microorganisms are becoming resistant to existing disinfection techniques involving biocides, ultraviolet light and heat treatment. In addition, some species of bacteria such as Bacillus subtilis, produce colonies or spores which agglomerate in clusters. Chlorination destroys such microorganisms on the surface but often leaves the innermost intact. Clusters or flocs of fine particles (e.g. clay) can entrap bacteria which also protects them against chlorination. Ultrasound can offer effective means of declumping as well as killing bacteria.
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 place 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 to as a method of counting or estimating 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.
We use 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.
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 ultrasonic 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 variables that may alter the effectiveness of ultrasonic on bacteria are:
- The frequency of the ultrasound waves
- The amplitude of ultrasound waves
- The exposure time
- The type of bacteria
- Bacteria medium (liquid, solids)
However at school level, your independent variable is exposure to any available ultrasonic (Possible values are Yes, No)
Dependent variable is the bacteria count.
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 ultrasonic can disinfect and kill bacteria. My hypothesis is based on my gathered information and the fact that high amplitude ultrasound waves create localized heat caused by vibration of molecules.
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 ultrasonic be able to disinfect (kill bacteria or disrupt the growth or reproduction of bacteria), we will have a lower bacteria count on samples exposed to ultrasonic for a period of time. For your experiment, get a sample containing bacteria and divide it in two parts. Expose one part to ultrasonic 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.
Step 1:
Prepare 24 culture media plates for growing bacteria. You may purchase a bacteria culture kit and prepare your plates using nutrient agar that comes in the kit. Click here to See sample kit.
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. Do the following steps.
- Prepare 1:10 dilution of the sample. To do this, take 20 mL of the sample and blend it with 180 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
- Let it cool and use it to spread dilution around the plate
- Do this on six plates for each of the four different dilutions. (skip 1:10 dilution. It usually have too many bacteria and is not countable.)
- Place 3 plates of each group in a glass or metal container with a working ultrasonic transducer for one hour. Mark these plates.
- incubate all plates at 37 degrees Celsius for 24 hours and then count the bacterial colonies.
You can repeat the above procedures while changing the ultrasonic source and/ or exposure time.
Record the results in a table like this:
| Dilution | Bacteria count for group exposed to ultrasonic | Bacteria count for groups NOT exposed to ultrasonic |
| 1:100 plate 1 | ||
| 1:100 plate 2 | ||
| 1:100 plate 3 | ||
| 1:100 Average | ||
| 1:1000 plate 1 | ||
| 1:1000 plate 2 | ||
| 1:1000 plate 3 | ||
| 1:1000 Average | ||
| 1:10,000 Plate 1 | ||
| 1:10,000 Plate 2 | ||
| 1:10,000 Plate 3 | ||
| 1:10,000 Average | ||
| 1:100,000 Plate 1 | ||
| 1:100,000 Plate 2 | ||
| 1:100,000 Plate 3 | ||
| 1:100,000 Average | ||
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.
Materials and Equipment:
- Ultrasonic mouse repellent
- 10 test-tubes of sterilized water
- 24 PCA plates
- blender
- Bunsen burner
- graduated cylinder
- Ethanol
- glass hockey stick
- pipettes
- refrigerator
- incubator (Any warm room or cabinet or oven)
- microwave
- scale
- large beaker
- hot plate
Note: List of material depends on your final procedure design. You may modify these procedures based on what is available to you. Ultrasonic mouse repellent may be purchased from hardware stores. You may be able to substitute it with any other high amplitude ultrasonic device with frequency about 20 kilo hertz.
Where do I buy bacteria?
Bacteria are everywhere. The experiments suggested in this project guide do not require any specific type of bacteria. General household bacteria or bacteria from any polluted water may be used. If you need to purchase any specific bacteria you may search the Internet for Bacteria Culture and find websites such as wardsci.com where you can order bacteria online.
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:
Since you are diluting your sample, at the end you need to calculate the bacteria count per gram or per mL of your sample.
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.
References:
List of References
Q. What is the difference between petri-dishes and nutrient agar plates? are these the same things?
A. Petri dishes are clear plastic dishes with a diameter of about 10cm. You usually buy them empty, in packs of 10 or 20.
Bacteria can not grow in empty dishes, so you make a warm solution of agar and some foods for bacteria and fill your petri dishes with this warm solution. It will become a gel as it cools.
A petri dish containing agar and food for bacteria is called a nutrient agar plate.
