Introduction: (Initial Observation)
Destroying microorganisms or controlling their growth is of considerable importance given that many microbes cause disease. Also, when preparing cultures for study, it is important that contamination be kept at a minimum. Sterilization is the process by which all living microorganisms are killed or removed from an inanimate object or living tissue. Disinfection is a less thorough process whereby microorganisms are destroyed, inhibited, or removed, usually from an inanimate object. A disinfected object is not necessarily sterile because some microorganisms or their endospores (dormant form of a microbe) may have survived.
Sanitization is third, even less thorough, process in which populations of microorganisms or inanimate objects are reduced to a level that is considered safe by public health standards.
Antimicrobials are agents used in sterilization, disinfection, and sanitization. Examples of antimicrobial agents include heat, gas, radiation, and chemicals. Two types of chemical microbial agents are disinfectants and antiseptics. Disinfectants are typically used on inanimate surfaces such as floors, workbenches and lab equipment. Antiseptics are used on living tissues such as human skin or mucous membranes.
Information Gathering:
Find out about antiseptics and soaps. Read books, magazines or ask professionals who might know in order to learn about the effects of antiseptics and soaps on bacteria. Keep track of where you got your information from. Following are some information that you may use.
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.
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.
Antiseptics and Disinfectants
Antiseptics are for use on people.
Disinfectants are for use on objects and surfaces.
Antiseptics
Antiseptics are used for:
- Skin, cervical, or vaginal preparation before a clinical procedure
- Surgical scrub
- Handwashing in high-risk situations, such as before an invasive procedure or contact with a client at high risk of infection (e.g., a newborn or immunosuppressed client)
Antiseptics are not meant to be used on inanimate objects, such as instruments and surfaces. Antiseptics are designed to be used for reducing or destroying microorganisms on the skin or mucous membranes without damaging these tissues. They usually do not have the same killing power as chemicals used for high-level disinfection of inanimate objects. Therefore, antiseptic solutions should never be used to disinfect inanimate objects, such as instruments and reusable gloves. In addition, items such as pickup forceps, scissors, scalpel blades, and suture needles should never be left soaking in an antiseptic solution.
Note:
- Chlorhexidine (with or without cetrimide) and iodophor solutions are the preferred antiseptics for use in health care settings. While products containing chlorhexidine are ideal for surgical handscrub and skin preparation in general, they may not be the best antiseptics to use in the genital area because of the small potential for irritation. Iodophors are a better choice for use in the genital area; however, if an iodophor is not available, a product containing chlorhexidine is the best alternative.
- Hexachlorophene and iodine solutions are not recommended for use in routine surgical handscrub or for use on mucous membranes.
- Benzalkonium chloride (BZK) and mercury laurel are disinfectants and should not be used as antiseptics.
Alcohol
Alcohol is a disinfectant for INTACT skin or inanimate objects. Use it to clean gear such as splinter forceps. It can also be used to clean skin around (not inside) a wound. Individual pads are more practical than a bottle.
Alcohol should NOT be used inside wounds or on open wounds. When used on exposed tissue, alcohol kills some of your tissues along with the germs. This delays healing. When used INSIDE a wound, alcohol can actually make the wound MORE infection prone by turning your tissue into dead “germ food.”
Antiseptics
Antiseptics are substances that kill bacteria and other micro-organisms. Many are mild enough to be applied to the skin to cleanse wounds, or to be taken internally (this distinguishes them from some disinfectants, which are more powerful). The discovery of antiseptics to disinfect wounds was made by Joseph Lister in the 1860s, and dramatically reduced the death rate during amputations. He used a solution of carbolic acid (phenol) which, along with other phenolic compounds, is still one of the main antiseptic agents today. Other common antiseptics include iodine, boric acid and ethanol.
The History of Iodine and Povidone-lodine
Iodine has long been accepted as a uniquely effective antiseptic and used widely both for the prevention and treatment of infection. Its popularity as an anti-microbial against the complete pathogenic spectrum has, however, been limited by a number of undesirable factors.
Many solutions of iodine and its salts were formulated to overcome these problems. Lugol’s solution, Mandell’s paint and tincture of iodine are still widely used, but do little to enhance the acceptability of iodine.
It was discovered, however, in the early 1950’s that when iodine is complexed with the inert polymer, polyvinylpyrrolidone (povidone), it ceases to irritate, sensitize or stain, and yet retains its unique microbicidal activity.
The removal of the undesirable side effects of iodine has allowed this broad spectrum antiseptic to be more widely used than every before in the form of povidone-iodine.
This complex of iodine has been formulated into a wide variety of presentations for use in hospitals and general practice.
How Iodine Works as a Microbicide
Iodine is an extremely effective, quick acting, total microbicide. To have a anti-microbial effect iodine (I2) needs to be activated by water (H20) to form the active H20I+.
Iodine must be in solution to work. (Dry powder formulations rely on tissue water).
Microbicidal Activity of Iodine
The microbicidal effectiveness of elemental iodine is based on two factors:-
a) Its marked oxidising effect on the amino acid components of respiratory enzymes found in the membranes of micro organisms. Micro organisms have many respiratory enzymes e.g. tyrosinase located on their cell membranes. When iodine comes into contact with them the cell membrane disrupts and kills the organism.
Side Effects of Iodine
* Irritation and Sensitivity Reactions
Iodine can cause irritation and sensitivity reactions in a small number of people due to toxic reactions following application of too high a concentration to the skin. Tincture of iodine contains 3,000 parts per million (ppm) free iodine.
Iodine reactions are very rare occurring in only 0.7% of atopic (allergic) subjects. (Less than 1 in 1000 of the general population)
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 is an example:
The purpose of this experiment is to determine which antiseptic works best against household bacteria.
I became interested in this idea when I was on medication for a sickness and noticed how much the medication costs and its side effects. The information gained from this experiment might prevent people from getting sick if household detergents and antiseptics can kill sickness-causing bacteria around the house. It could also prevent society from wasting time and money for doctor visits, buying prescription medicine and paying medical bills.
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.
Independent variable: different antiseptics and disinfectants.
Dependent variable: how much growth is inhibited (area around the disks where
no growth has occurred).
Control: sterile disk with sterile water.
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.
Hypothesis example 1:
Various antiseptics and disinfectants will inhibit bacterial growth to varying degrees.
Hypothesis example 2:
My hypothesis is that isopropyl alcohol will work better than ammonia and Lysol. I base my hypothesis on the fact that isopropyl alcohol is used in hospitals and doctors’ offices for antiseptic purposes before administrating injections.
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:
Introduction: In this experiment you will try to grow bacteria on the surface of nutrient agar gel in a petri dish. You will also place small paper disks saturated with different antiseptics on the surface of the agar plate. After about 3 days in an incubator (or warm cabinet) you should be able to see how effective each antiseptic is. When an antiseptic is more effective, the bacteria free area around it is larger.
With this experiment you can see the relative effectiveness of some disinfectants and antiseptics on some household bacteria. Steps include:
- Preparation of a clear agar gel with nutrients such as chicken broth
- pouring the hot liquid gel in petri dishes and let them cool off and solidify.
- Streak the plates with bacteria.
- Place paper disks saturated with antiseptics on the plates.
- Place the plates in an incubator and observe them after3 days.
Materials:
- 6 nutrient agar plates. (Make them at home or buy TSA plates. TSA stands for Tryptic Soy Agar). If you need to make them at home, you will need agar and chicken broth.
Click here to learn more about bacteria culture media… (optional) - Sterile swabs
- Bacteria culture. (You may buy known bacteria such as E. coli or you may use household bacteria found in your kitchen sink.)
- Test tube racks
- Sterile disks. (Make them yourself by cutting filter paper or by punching filter paper using a paper puncher)
- Forceps
- Bunsen burner or alcohol burner
Various solutions:
- ethanol (disinfectant)
- antibacterial hand soap (antiseptic)
- iodine (antiseptic)
- dishwasher detergent (disinfectant)
- powdered hand soap from Haworth bathrooms (antiseptic)
- sterile water (control)
Also recommended Biohazard bags if you are buying bacteria (Bio hazard bags are white plastic bags printed and marked as bio hazards. The idea is that when you dispose your contaminated materials such as petri dishes and test tubes, someone may think of them as reusable and pick them up without necessary care. A bio hazard bag has the necessary warning to prevent such incidents. For additional safety, I suggest to sterile everything in high heat of an autoclave or oven prior to disposal. If you don’t have bio hazard bags, write your own warnings on white bags prior to disposal.)
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. You can also purchase such materials from MiniScience.com online store or from klk.com.
Procedure:
- Prepare 6 nutrient agar plates and one pack of sterile swabs. Click here to learn how you can prepare your own plates.
- Label each of the 6 plates with the following abbreviations.
Ethanol=EtOH
dishwasher detergent=DD
antibacterial hand soap=AHS
Iodine=I
powder hand soap=PHS
control=C - Prepare your household bacteria culture. To do that use a swab to remove some bacteria from the drain area of the kitchen sink and dissolve it in water in a test tube. Alternatively you may remove bacteria from your mouth before brushing.
- Dip a clean swab into the bacteria culture, taking only a small amount. Distribute the sample over the plate as is illustrated below.
Rotate plate 90 degrees and repeat.
Rotate 45 degrees and perform same swab.
In this way you will cover the entire surface of the agar plate.
5. Replace the top on the plate, and dispose of the swab in the small biohazard bag.
6. Repeat the streaking plate process for the second nutrient agar plate, using a different swab.
7. Obtain forceps, alcohol, and sterile disks.
8. Put a small amount of alcohol on the tip of the forceps, and pass it through the flame on the Bunsen burner, allowing the alcohol to burn off.
9. With the now sterile forceps, gently remove a sterile disk from the container.
10. Using a sterile pipette, transfer 1 drop of iodine solution on the disk. Dispose of the pipette appropriately.
11. Place the disk it in the center of one of the 6 nutrient agar plates.
12. Repeat this process for the remaining 5 plates, placing one disk, saturated with one specific antiseptic in each plate. Put the lids for all plates immediately.
13. Incubate plates at 37º C for 48 hours.
14. You should observe that some or all of the disks are surrounded by a region with no bacterial growth. Measure the diameter of these regions, and record your results in the result table.
15. Dispose of your plates in the biohazard bag, wash your hands, and wipe down lab bench with alcohol.
Note 1: In the above procedures we are suggesting to use each nutrient agar plate with one antiseptic disk. With some more care, it is possible to test more than one antiseptic in one nutrient agar plate (as shown in the picture above).
Note 2: This project was initially designed for students who may purchase ready made nutrient agar plates known as TSA plates and purchase E.Coli bacteria culture. We have now modified this so students don’t have to purchase ready made plates and bacteria culture.
Results
Results table: Affect of antiseptics and disinfectants on bacterial growth.
| Treatment | Area of no growth (mm) | Other observations |
| EtOH | ||
| AHS | ||
| I | ||
| DD | ||
| PHS | ||
| C |
- Did the results support or falsify your hypothesis or predictions?
- Interpret your results. What do your results mean?
- What were some weaknesses in your experiment, and how could you improve upon them?
- After completing your experiment and its interpretation, what are some new hypotheses you could test related to what you found?
Another Sample Procedure
1. Gather all materials needed.
2. Add bacteria to culture flask.
3. Let stand for 24 hours.
4. Label 1 test tube C.
5. Label four more test tubes: 2a, 2b, 2c and 2d.
6. Label four more test tubes: 3a, 3b, 3c and 3d.
7. Label the last four test tubes: 4a, 4b, 4c and 4d.
8. Add 50ml of bacteria infested water to each test tube.
9. Set the control test tube aside which will have no antiseptic added.
10. Measure out 5ml of ammonia and add it to flask 2a.
11. Measure out 10ml of ammonia and add it to flask 2b.
12. Measure out 15ml of ammonia and add it to flask 2c.
13. Measure out 20ml of ammonia and add it to flask 2d.
14. Let flasks stand for two minutes.
15. Label the Petri dishes the same as the test tubes.
16. Add 1ml of each tube contents to each Petri dish labeled the same.
17. Measure 5ml of isopropyl alcohol and add it to flask 3a.
18. Measure 10ml of isopropyl alcohol and add it to flask 3b.
19. Measure 15ml of isopropyl alcohol and add it to flask 3c.
20. Measure 20ml of isopropyl alcohol and add it to flask 3d.
21. Let flasks stand for two minutes.
22. Add 1ml of each tube contents to each Petri dish labeled the same.
23. Measure 1ml of Lysol and add it to flask 4a.
24. Measure 5ml of Lysol and add it to flask 4b.
25. Measure 10ml of Lysol and add it to flask 4c.
26. Measure 20ml of Lysol and add it to flask 4d.
27. Let flasks stand for two minutes
28. Add 1ml of each tube contents to each Petri dish labeled the same.
29. Set incubator to 34 degrees Celsius.
30. Place all Petri dishes into the incubator.
31. Observe 72 hours from incubating and proceed with a colony count under a magnifier glass or microscope.
Materials and Equipment:
List of material depends on your final experiment design. Following is a sample.
Sample Materials List:
| QUANTITY | ITEM DESCRIPTION |
| 1 | Bacteria culture (Lactobacillus) |
| 16 | Petri dishes preloaded with agar |
| 16 | Test tubes |
| 1 | Bottle of isopropyl (rubbing) alcohol (70%) |
| 1 | Bottle of ammonia |
| 1 | Bottle of Lysol |
| 1 | Box of rubber gloves |
| 1 | Pencil |
| 1 | Pad of paper |
| 2 | Hours to conduct the experiment |
| 1 | Computer |
| 1 | Chair |
| 1 | Microscope |
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.
Sample Results
The original purpose of this experiment was to determine which antiseptic works best against specific bacteria. The isopropyl alcohol did not even affect the bacteria at 5ml, disinfected 1/3 of them in the 10ml and totally killed them in the 15ml and 20ml. The ammonia proved to be less affective and barely disinfected at all. 5ml and 10ml didn’t work at all, 15 and 20ml did but almost too small to notice. Lysol was the most effective even after being deluded to the recommended concentration it disinfected the samples entirely in all of the amounts
Calculations:
No calculation is required for this project.
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.
Sample Research Report
Bacteria are microorganisms that lack nuclear membranes. Scientists believe they are the oldest living things on earth. Bacteria are less than one micron in length (.0001 mm). Hundreds of thousands of bacteria can fit in the period at the end of this sentence. Bacteria can multiply at an incredible rate if they can get enough food. Within a few days, multimillion colonies can be seen with ease without the aid of a microscope.
Classification
There are two distinct kinds of life: procaryotic and eucaryotic. Procaryotes are things that are microscopic and do not have internal membranes. Eucaryotes are macro or microscopic and have internal compartments.
Bacteria are also classified by shape. If the shape is spherical, it is called a cocci. A rod-shaped bacteria is a bacilli and a corkscrew is called a spirochetes.
Bacteria are also be classified by whether they need oxygen. Bacteria that need oxygen to survive are aerobic bacteria and those that do not need oxygen are anaerobes.
One last way of classifying bacteria is by the method used to obtain carbon and energy. Autotrophs obtain their carbon from carbon dioxide and their energy from different things. Photoautrotrophs get their energy from light and use the process called photosynthesis for their energy. Photosynthesis uses light to change substances into matter on which the bacteria can feed. The remaining bacteria are called heterotrophs. They receive their carbon from eating organic molecules. There is a subspecies called photoheterotrophs, which obtain their energy from the sun.
History
In the later part of the 1600’s, Antoni van Leeuwenhoek was the first person to study bacteria. He became the inventor of the microscope. Other great microbiologists of the past include Robert Koch, the person who showed us some bacteria can cause disease; and Selman Waksman, who found a large variety of soil bacteria and who also produced antibiotics that completely revolutionized the way we treat bacteria and disease.
Structure
Like all cells, bacteria contain DNA, but unlike most cells it is in a circular pattern instead the normal helix shape. Many bacteria have small limbs extending from their membranes called pilus. Pilus are hair-like teeth that can attach to rocks, prey and many other things. Other have extensions called flagella. They use these extensions to propel themselves toward nutrients.
Reproduction
Bacteria reproduce by a process known as binary fission. They basically split into two separate life forms. When these two new bacteria reach full size and maturity, they again split and so the process continues.
How Bacteria Affect Our Daily Lives
Bacteria cover almost every natural surface known to man and live inside many plants and animals. Bacteria are very important in the survival of the ocean ecosystem. Bacteria are at the bottom of the food chain. They are eaten by plankton, which is eaten by larger animals, which are, in turn, eaten, etc. Bacteria are also important removers of waste. They feed on dead and decaying animals and other wasters, turning them into valuable soil.
History of Soap
Soap is a cleaning agent that is made of animal and plant fats and the sodium or potassium salt in the fatty acid, which is formed by the interaction of the fats and alkali. The first soap was talked about in the Old Testament. This was not true soap but was made from just tree bark ashes alone. The 1200’s introduced the soap industry to France. Most soap was made of goat tallow with beech ash. The French then found how to made soap out of olive oil.
Summary
Bacteria are a small but important part of our world. Scientists believe they have been on Earth longer than any other life form. The means to control disease-causing bacteria is essential for the health of mankind but a balance must be struck. Bacteria as a life form must never be threatened because they are one of the basic corner stones on which the delicate balance of nature is built. Without bacteria, life as we know it would cease to exist.
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.
Sample CONCLUSION
My hypothesis is that isopropyl alcohol will work better than ammonia and Lysol.
The results indicate that this hypothesis should be rejected because the Lysol worked far better then the ammonia and the alcohol.
Because of the results of the experiment I wonder if the company that manufactures Lysol should create a product that hospitals could use on patients instead of isopropyl alcohol.
If I were to conduct this project again I would use antibacterial soap in my experiment and I also would have been more prepared for the scientific committee. I would of also of used two strands of bacteria to see if my results are more widely occurring.
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 (Bibliography)
“Bacteria” Macropedia Britannica, (1988).
“Bacteria” Microsoft Encarta, (1998).
“Bacteria” Microsoft Encarta, (1999).
Carter, Joseph, et al. Life Science Ginn and Company, (1971).
“Detergents” Microsoft Encarta, (1999).
Dravistion, Michael. Microbiology Warner Books. (1987).
“Soap” Microsoft Encarta, (1999).
Schessinger, David. “Bacteria” World Book Encyclopedia. (1994)
