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Science Projects > Chemistry Projects > How To Grow Bacteria and More  

How To Grow Bacteria and More

If learning how to grow bacteria in a petri dish interests you, read on.

How Can Bacteria Help Us? How Can Bacteria Harm Us? What Are Antibacterial Agents? Experiment #1: Cheek Cell Swab Experiment #2: Testing Antibacterial Agents Experiment #3: Soap Survey Experiment #4: Bacteria in the Air Experiment #5: Homemade Yogurt More Experiment Ideas

Bacteria Overview

Bacteria are one-celled, or unicellular, microorganisms . They are different from plant and animal cells because they don’t have a distinct, membrane-enclosed nucleus containing genetic material. Instead, their DNA floats in a tangle inside the cell.

Individual bacteria can only be seen with a microscope, but they reproduce so rapidly that they often form colonies that we can see. Bacteria reproduce when one cell splits into two cells through a process called binary fission. Fission occurs rapidly in as little as 20 minutes. Under perfect conditions a single bacterium could grow into over one billion bacteria in only 10 hours! (It’s a good thing natural conditions are rarely perfect, or the earth would be buried in bacteria!)

Growing and testing bacteria is a fun any-time project or a great science fair project. Bacteria are everywhere, and since they reproduce rapidly they are easy to study with just a few simple materials. All you need are some petri dishes , agar, and sterile swabs or an inoculating needle . Agar is a gelatinous medium that provides nutrients and a stable, controlled environment for bacteria growth . Most bacteria will grow well using nutrient agar , but some more fastidious bacteria (those with more complex nutrient requirements like Bacillus stearothermophilus , Branhamella catarrhalis , and Bacillus coagulans ) prefer tryptic soy agar .

You also need a source for bacteria, and this is not hard to find! You can swab your mouth or skin, pets, soil, or household surfaces like the kitchen sink or toilet bowl. If you want to study a particular type of bacteria, you can also purchase live cultures . Keep reading to see four experiments using bacteria, and many more ideas for science projects (also consider this hands-on Bacteria Growing Kit )! Adult supervision is recommended when working with bacteria.

How Can Bacteria Help Us?

Where would we be without bacteria? Well, we might not be getting bacterial diseases, but we would still be a lot worse off! Bacteria perform all sorts of very important functions, both in our bodies and in the world around us. Here are just a few.

Digestion. Our large intestines are full of beneficial bacteria that break down food that our bodies can’t digest on their own. Once the bacteria break it down, our intestines are able to absorb it, giving us more nutrients from our food.

Vitamins. Bacteria in our intestines actually produce and secrete vitamins that are important for our health! For example, E. coli bacteria in our intestines are a major source of vitamin K. (Most E. coli is good for us, but there is a harmful type that causes food poisoning.)

Food. Bacteria are used to turn milk into yogurt, cheese, and other dairy products.

Oxygen. Cyanobacteria (which used to be called blue-green algae) live in water and perform photosynthesis, which results in the production of much of the oxygen we need to breathe.

Cleanup. Oil spills, sewage, industrial waste — bacteria can help us clean all of these up! They ‘eat’ the oil or toxins and convert them into less harmful substances.

Bacteria are amazing creatures, aren’t they? They can be so dangerous and yet so important at the same time. Keep reading to see an experiment that uses good bacteria!

How Can Bacteria Harm Us?

Some types of bacteria cause disease and sickness. These kinds of bacteria are called pathogens. They reproduce very rapidly, like all bacteria. These come in many forms and can cause illnesses from an ear infection to strep throat to cholera. They can get into our bodies via our mouth and nose, or through cuts and scrapes. Some are airborne, others are found in food, resulting in food poisoning. Bacteria are also the cause of plaque buildup on our teeth, which can lead to cavities and gum disease.

Before the discovery of antibiotics, many severe bacterial diseases had no cure and usually resulted in death. Antibiotics work by destroying bacteria or inhibiting their reproduction while leaving the body’s own cells unharmed. After a time, some bacteria develop resistance to an antibiotic, and it will no longer be effective against them. Because of this, scientists are always researching new antibiotics. (Many diseases, such as chicken pox, hepatitis, or polio, are caused by viruses rather than bacteria. Antibiotics have no effect against these diseases.)

Bacterial infections are common, but many of them can be avoided by good cooking, cleaning, and hand-washing practices.

What Are Antibacterial Agents?

How do people stop bacteria from growing and spreading? They control it in two ways: by killing the bacteria cells, and by stopping the bacteria from reproducing. An agent is a solution or method which either kills or stops reproduction. Bactericides are agents that kill bacteria cells. Static agents inhibit cell growth and reproduction.

There are a variety of ways to kill bacteria or keep it from reproducing.

Physical methods:

• Sterilization. The application of heat to kill bacteria. Includes incineration (burning), boiling, and cooking.
• Pasteurization. The use of mild heat to reduce the number of bacteria in a food.
• Cold temperatures. Refrigeration and freezing are two of the most common methods used in homes, for preserving food’s life span.

Chemical methods:

• Antiseptics. These agents can be applied directly to living tissues, including human skin.
• Disinfectants. These agents are not safe for live tissues. Disinfectants are used to clean toilets, sinks, floors, etc.
• Some food preservatives are: sodium benzoate, monosodium glutamate (MSG), sulfur dioxide, salts, sugar, and wood smoke.
• Amoxycillin and Ampicillin—inhibit steps in cell wall synthesis (building)
• Penicillin—inhibits steps in cell wall synthesis
• Erythromycin—inhibits RNA translation for protein synthesis

SAFETY NOTE

While most environmental bacteria are not harmful to healthy individuals, once concentrated in colonies, they can be hazardous.

To minimize risk, wear disposable gloves while handling bacteria, and thoroughly wash your hands before and after. Never eat or drink during bacteria studies, nor inhale or ingest growing cultures. Work in a draft-free room and reduce airflow as much as possible. Keep petri dishes with cultured mediums closed—preferably taped shut—unless sampling or disinfecting. Even then, remove the petri dish only enough to insert your implement or cover medium with bleach or 70% isopropyl alcohol.

When finished experimenting, seal dishes in a plastic bag and dispose. Cover accidental breaks or spills with bleach or alcohol for 10 minutes, then carefully sweep up, seal in a plastic bag, and discard.

Preparing Culture Dishes

Before you can grow bacteria, you’ll need to prepare sterile culture dishes. A 125ml bottle of nutrient agar contains enough to fill about 10 petri dishes.

Water Bath Method – Loosen the agar bottle cap, but do not remove it completely. Place the bottle in hot water at 170-190 °F until all of the agar is liquid. To prevent the bottle from tipping, keep the water level even with the agar level.

• Let the agar cool to 110-120 °F (when the bottle still feels warm but not too hot to touch) before pouring into petri dishes.
• Slide open the cover of the petri dish just enough to pour agar into the dish. Pour enough agar to cover 1/2 to 2/3 of the bottom of the dish (about 10-13ml). Don’t let the bottle mouth touch the dish. Cover the dish immediately to prevent contamination and tilt it back and forth gently until the agar coats the entire bottom of the dish. (Fill as many dishes as you have agar for: you can store extras upside down until you’re ready to use them.)
• Let the petri dishes stand one hour for the agar to solidify before using them.

Experiment #1: Cheek Cell Swab

Make a culture dish using the instructions above. Once the culture dish is prepared, use a sterile cotton swab or inoculating needle and swab the inside of your cheek. Very gently rub the swab over the agar in a few zigzag strokes and replace the lid on the dish. You’ll need to let the dish sit in a warm area for 3-7 days before bacteria growth appears. Record the growth each day with a drawing and a written description. The individual bacteria are too tiny to see without a high-power microscope, but you can see bacteria colonies. Distinguish between different types of bacteria by the color and shape of the colonies.

Experiment #2: Testing Antibacterial Agents

Preparing Sensitivity Squares

One method for testing the antibacterial effectiveness of a substance is to use ‘sensitivity squares.’ Cut small squares of blotter paper (or other absorbent paper) and then soak them in whatever substance you want to test: iodine, ethyl alcohol, antibacterial soap, antiseptics, garlic, etc. Use clean tweezers to handle the squares so you don’t contaminate them. Label them with permanent ink, soak them in the chosen substance, and blot the excess liquid with a paper towel.

Collecting Bacteria

Decide on a source for collecting bacteria. For using sensitivity squares, make sure there is just one source, and keep each dish as consistent as possible. Sources could include a kitchen sink, bathroom counter, cell phone, or another surface you would like to test. Rub a sterile swab across the chosen surface, and then lightly rub it across the prepared agar dish in a zigzag pattern. Rotate the dish and repeat.

Setting Up an Experiment

Each experiment should have a control dish that shows bacteria growth under normal conditions and one or more test dishes in which you change certain variables and examine the results. Examples of variables to test are temperature or the presence of antiseptics. How do these affect bacteria growth?

• Label one dish ‘Control.’ Then in your test dish, use tweezers to add the sensitivity squares that have been soaked in a substance you wish to test for antibacterial properties. It’s a good idea to add a plain square of blotter paper to see if the paper by itself has any effect on bacteria growth. For best results, use multiple test dishes and control the variables so the conditions are identical for each dish: bacteria collected from the same place, exposed to the same amount of antibacterial substance, stored at the same temperature, etc. The more tests you perform, the more data you will collect, and the more confident you can be about your conclusions.
• Place all the dishes in a dark, room-temperature place like a closet.

Wait 3-7 days and examine the bacteria growth in the dishes, without removing the lids. You will see multiple round dots of growth; these are bacteria colonies. Depending on where you collected your bacteria samples, you may have several types of bacteria (and even some mold!) growing in your dishes. Different types of colonies will have different colors and textures. If you have a compound or stereo microscope, try looking at the colonies up close to see more of the differences.

Compare the amount of bacteria in the control dish to the amount in the test dishes. Next, compare the amount of bacteria growth around each paper square. Which one has bacteria growing closest to it? Which one has the least amount of bacteria growing near it? If you did more than one test dish, are the results similar in all the test dishes? If not, what variables do you think might have caused the results to be different? How does this affect your conclusions?

For a variation on this experiment, test the effect of temperature on bacterial growth instead of using sensitivity squares. Put a control dish at room temperature, and place other dishes in dark areas with different temperatures.

Experiment #3: Soap Survey

Every time you touch something you are probably picking up new bacteria and leaving some behind. This is how many infectious diseases spread — we share our bacteria with everyone around us! Even bacteria that lives safely on our skin can make us sick if it gets inside our bodies through our mouths or cuts and scrapes. This is one reason why it is so important that we wash our hands frequently and well.

What kind of soap works best for cutting down on the bacteria on our hands? You can test this by growing some bacteria cultures using agar and petri dishes.

• Two (or more)  petri dishes
• Sterile swabs
• Blotter paper  or other absorbent paper
• Forceps  or tweezers
• Different kinds of hand cleaners: regular soap, antibacterial soap, dish soap, hand sanitizer

1. Prepare the agar according to the directions on the label, then pour enough to cover the bottom of each petri dish. Cover the dishes and let them stand for about an hour until the agar has solidified again. (If you aren’t going to use them right away after they have cooled, store them upside down in the refrigerator.)

2. When your petri dishes are ready, collect some bacteria from your hand or the hand of a volunteer. (Make sure the person hasn’t washed his or her hands too recently!) Do this by rubbing the sterile swab over the palm in a zigzag pattern.

3. Remove the cover from the petri dish and lightly rub the swab back and forth in a zigzag pattern on the agar. Turn the dish a quarter turn and zigzag again. Cover the dish and repeat steps two and three for the other dish, using a new sterile swab. Label the dishes “Test” and “Control.” (You may want to do more than one test dish, so you can compare the results.)

4. Cut the blotter paper into small “sensitivity squares.” Use permanent ink to label the squares for the different types of hand cleaners you are going to test, e.g., “R” for regular soap, “A” for antibacterial soap, and “S” for hand sanitizer. Using tweezers, dip each square into the appropriate cleaner. Blot the excess cleaner on a paper towel and then place the squares on the agar in the “Test” dish. (Spread the squares out so there is distance between them.) Add one square of plain blotter paper to test if blotter paper by itself has any effect. Don’t put any squares in the “Control” dish – this one will show you what the bacterial growth will look like without any soap.

5. Put the dishes in a dark, room-temperature place like a closet and leave them undisturbed for a few days.

What Happened

The rate of bacteria growth in your dishes will depend on temperature and other factors. Check your cultures after a couple of days, but you’ll probably want to wait 5-7 days before recording your data. You will see multiple round dots of growth; these are bacteria colonies. There may be several types of bacteria growing in the dishes. Different types of colonies will have different colors and textures.

For each soap test, count and record the number of bacteria colonies in each dish. To see how effective each soap was, divide the number of colonies in the test dish by the number of colonies in the control dish, then subtract the result from 1 and write the answer as a percentage. For example, if your control dish had 100 colonies and your soap test dish had 30, the soap eliminated 70% of the bacteria: 1 — (30 ÷ 100) = .7 = 70%

According to your results, which type of soap was the most effective at eliminating bacteria?  Does “antibacterial” soap really work better than regular soap? How well did washing hands in water without soap work? What further tests could you do to determine which soaps and hand washing methods are most effective at eliminating bacteria?

Experiment #4: Bacteria in the Air

You need two culture dishes for this experiment, in which you’ll demonstrate how antibacterial agents (such as antibiotics and household cleaners) affect bacteria growth.

Leave the dishes with their lids off in a room-temperature location. Leave the culture dishes exposed for about an hour.

While you wait, cut small squares of paper (blotter paper works well), label them with the names of the antibacterials you’re going to test (e.g. ‘L’ for Lysol, ‘A’ for alcohol, etc.), and soak each in a different household chemical that you wish to test for antibacterial properties. If you have time, you might also experiment with natural antibacterial agents, such as tea tree oil or red pepper. Wipe off any excess liquid and use tweezers to set each of the squares on a different spot in one of the culture dishes. The second culture dish is your ‘control.’ It will show you what an air bacteria culture looks like without any chemical agents.

Store the dishes (with lids on) in a dark place like a closet where they will be undisturbed for a few days. After 3-7 days, take both culture dishes and carefully observe the bacteria growth in each dish, leaving the lids on. The bacteria will be visible in small, colored clusters. Take notes of your observations and make drawings. You could also answer the following questions. In the control culture, How much of the dish is covered with bacteria? In the sensitivity square test culture, Have the bacteria covered this dish to the same extent as the control culture? What effect have each of the chemicals had on the bacteria growth? Did a particular chemical kill the bacteria or just inhibit its growth?

• For further study you could use an  antibiotic disc set  to see what different antibiotics can do against bacteria.
• For a  more advanced project , learn how gram staining relates to the use of antibiotics.

Experiment #5: Homemade Yogurt

Generally when people think of ‘bacteria,’ they think of harmful germs. However, not all forms of bacteria are bad! You can enjoy a tasty product of good bacteria by making a batch of yogurt at home.

You’ll need to use a starter (available at grocery or health food stores), or else one cup of plain, unflavored yogurt that has live cultures in it. (If it contains live cultures, it will say so on the container.)

Slowly heat four cups of milk until it is hot, but not boiling or scalding. The temperature should be around 95-120 degrees to kill some of the harmful bacteria. Cool slightly, until milk is warm, and then add one cup of active yogurt or the starter.

Put the mixture in a large bowl (or glass jars) and cover. Make sure that the bowl or jars are sterilized before using by either running them through the dishwasher or washing them with very hot water.

There are two different methods for culturing the yogurt mixture: You can put the covered bowl or jars into a clean plastic cooler, and fill the cooler with hot water to just below the top of the culture containers. With this method, you will need to occasionally refill the cooler with hot water, so that the temperature of the yogurt stays consistent. The other method is to wrap the containers in a heating pad and towels, setting the heating pad on low to medium heat.

Check the mixture after heating for 3 1/2 to 4 hours. It should be ‘set up,’ having a smooth, creamy consistency similar to store-bought yogurt. If the mixture is not set up yet, heat it for another 1-2 hours. When it is the right consistency, add some flavoring—such as vanilla extract, chocolate syrup, or berries—and store the yogurt in the refrigerator. It should keep for a couple of weeks. For safety, we suggest that you do not eat any yogurt that has separated or has a non-typical consistency.

More Bacteria Experiment Ideas

Here are some other project ideas for you to try on your own or use as a basis for a bacteria science fair project:

• Mouthwash . Swab your teeth and gums and see how well toothpaste or mouthwash work against the plaque-causing bacteria on your teeth.
• Dog’s mouth : Have you heard people say that dogs’ mouths are cleaner than humans’? Design an experiment to test whether this is really true!
• Band-aid . Some band-aids are advertised as being antibacterial. Test to see if they really work better than regular band-aids at inhibiting bacteria.
• Water bottle . Is it safe to keep refilling a water bottle without washing it? Test a sample of water from the bottom of a water bottle that has been used for a couple days and compare it to a sample from a freshly-opened, clean water bottle. You can also test to see if a bottle gets more bacteria in it if you drink with your mouth or with a straw.
• Shoes . Do bacteria grow in your shoes? Is there a difference in bacteria growth between fabric shoes and leather? Do foot powders work to cut down on bacteria?
• Toothbrush . Do bacteria grow on your toothbrush? What are some ways you could try to keep it clean? Mouthwash? Hot water?
• Makeup . Some people recommend getting new mascara every six weeks because bacteria can grow in the tube. Test this by comparing bacteria growth from old mascara and new, unused mascara. You can also test how much bacteria is on other kinds of makeup.

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How to Grow Bacteria in a Petri Dish

Last Updated: October 16, 2024 Fact Checked

This article was co-authored by Meredith Juncker, PhD . Meredith Juncker is a PhD candidate in Biochemistry and Molecular Biology at Louisiana State University Health Sciences Center. Her studies are focused on proteins and neurodegenerative diseases. This article has been fact-checked, ensuring the accuracy of any cited facts and confirming the authority of its sources. This article has been viewed 698,812 times.

Ever wanted to grow bacteria for a science project or just for fun? It's surprisingly simple! All you need is some agar (a special gelatinous growing material), a number of sterilized Petri dishes, and some disgustingly good sources of bacteria!

Preparing the Petri Dishes

• The easiest type of agar to use in this experiment is a nutrient agar which comes in powder form. You could also use any type of powder agar or any agar in general. You will need as much agar as you need, but don't use less than 1.2 grams (½ teaspoon) of agar powder for every 10 centimetres (3.9 in) Petri dish you wish to use.
• In a heatproof dish or bowl, stir 1.2 grams (½ teaspoon) of the nutrient agar powder into 60 millilitres (0.25  c) of hot water. Multiply these quantities by however many Petri dishes you plan on using.
• Place the bowl or dish in the microwave and let it begin to boil for 1 or more minutes, watching to make sure that the agar solution doesn't boil over.
• When the solution is ready, the agar powder should be completely dissolved and the liquid should be clear in color.
• Allow the agar solution to cool for several minutes before proceeding. You don't want to get burnt!

• Petri dishes must be completely sterilized before they are used for growing bacteria, otherwise, the results of the experiment could be affected. Newly purchased Petri dishes should come pre-sterilized and sealed in plastic packaging.
• Remove the Petri dish from its packaging and separate the two halves. Very carefully, pour the warm agar solution into the bottom half of the Petri dish - just enough to form a layer over the bottom of the dish. Work in the presence of a candle with a tall flame or a Bunsen burner to keep contamination low.
• Quickly replace the top half of the Petri dish to prevent any airborne bacteria from contaminating the experiment. Set the Petri dishes aside for 30 minutes to 2 hours, until the agar solution cools and hardens (when it’s ready it will resemble set Jell-O).

• Storing the Petri dishes in the refrigerator prevents the water inside the dishes from evaporating (bacteria need a moist environment to grow). It also allows the surface of the agar to harden slightly, which prevents any tearing or gouging when you transfer your bacteria samples.
• When storing Petri dishes in the refrigerator, the dishes should be placed upside down. This helps to prevent any condensation on the lid from dropping down and disrupting the growing surface.
• Agar-filled Petri dishes will be kept in the refrigerator for as long as a couple of months. When you are ready to use them, remove them from the refrigerator and allow them to reach room temperature before introducing your samples.

Growing Bacteria

• Direct contact: This is when bacteria are transferred to the Petri dish using direct contact, i.e. touching the agar. One of the most common ways of doing this is to simply press your fingertip (either before or after washing your hands) lightly onto the surface of the agar. However, you could also try pressing a fingernail or the surface of an old coin into the agar or even placing a small hair or drop of milk into the dish. Use a sterile cotton swab if you have one available. Use your imagination!
• Sample collection : With this method, you can collect bacteria from almost any surface and transfer it to the Petri dish, all you need are some clean cotton swabs. Simply grab a swab and swipe it over any surface you can think of - the inside of your mouth, a door handle, the keys on your computer keyboard, or the buttons of your remote control - then use it to streak the surface of the agar (without tearing it). These places harbor a lot of bacteria and should provide some interesting (and disgusting) results in a couple of days.
• If you like, you can place more than one sample of bacteria in each Petri dish - all you need to do is divide the dish into quadrants (quarters) and swipe a different sample of bacteria in each of them.
• It is recommended that you keep one quadrant of the Petri dish free of bacteria samples to use as a control group. This allows you to know if the agar had been contaminated prior to the introduction of bacterial samples.

• Make sure to label each Petri dish with the source of the bacteria it contains, otherwise, you won't be able to tell which is which. You can do this using some tape and a marker.
• As an extra precaution, you can place each Petri dish in a zipper-lock bag. This will provide an extra layer of protection against any hazardous bacteria colonies that may develop, but will still allow you to view the contents of the Petri dish.

• The ideal temperature for growing bacteria is between 70 and 98 degrees F (20-37 degrees C). If necessary, you can place the Petri dishes in a cooler location, but the bacteria will grow a lot more slowly.
• Leave the bacteria to develop for 4-6 days, as this will give the cultures enough time to grow. Once the bacteria begins to grow, you may notice a smell coming from the dishes.

• Use a notebook to record your observations on the contents of each dish and perhaps come to a conclusion about which locations had the most bacteria.
• If you like, you can measure the daily growth of the bacteria colonies by using a felt-tip marker to trace a circle around each colony on the bottom of the Petri dish. After several days, you should have a collection of concentric rings on the bottom of each dish. This will help you count and record the data.

• Once you have introduced bacteria to the Petri dish, use a cotton swab to place a small drop of hand sanitizing gel, disinfectant soap, or household bleach into the center of the bacteria sample, then continue the experiment as normal.
• As the bacteria in the dish grows, you should see a ring or "halo" around the spot where you placed the antibacterial agent where no bacteria is growing. This is known as the "kill zone" (or more accurately, the "zone of inhibition").
• You can measure the effectiveness of different antibacterial agents by comparing the size of the kill zones in each Petri dish. The wider the kill zone, the more effective the antibacterial agent.

Safely Disposing of the Bacteria

• Although most of the bacteria you grow will not be hazardous, large bacteria colonies may pose more of a risk - so you will need to kill them before disposal using household bleach.
• Protect your hands from the bleach by wearing rubber gloves, protect your eyes with plastic goggles, and protect your clothes by wearing an apron.
• Cover open cuts with rubber gloves and avoid ingesting or breathing in the bacteria as it grows.

• Be very careful not to let any of the bleach touch your skin, as it will burn.
• Then place the disinfected Petri dish into a ziplock plastic bag and dispose of the bag in the trash.

Expert Q&A

• Try using a potato dextrose agar as a growth medium. Prepare a potato dextrose medium by boiling a 20 grams (0.71 oz) potato, 4 gm (2 tsp) of agar, and 2 gm (1 tsp) of dextrose in a beaker. Put this solution in a Petri dish and let it dry. Take some sterile cotton swabs and rub them over any place (remote, door handle, water pipe, etc.). Close the Petri dish using plastic wrap. Let it incubate for 24 hours in a warm place. The next day, check the Petri dish. You should be able to see colonies of bacteria. [9] X Research source Thanks Helpful 1 Not Helpful 0
• Petri dishes are not reusable. When left unattended the bacteria will multiply uncontrollably. Thanks Helpful 5 Not Helpful 1

Tips from our Readers

• You can make your own agar by combining ½ teaspoon of beef stock powder, ¼ cup of water, 1 teaspoon of sugar, and 1 teaspoon of gelatin.
• Make sure you do not grow bacteria above 40°F (4°C). This is considered the "danger zone" where dangerous bacteria can grow!

• Melted agar is very hot and sticks to your skin, meaning it could give you serious burns if touched. Thanks Helpful 12 Not Helpful 0
• Never put anything in the dish that is likely to grow into dangerous bacteria, such as body fluids. Thanks Helpful 0 Not Helpful 0
• If the dish is reopened it could cause serious illness. Thanks Helpful 0 Not Helpful 0

You Might Also Like

• ↑ https://learning-center.homesciencetools.com/article/bacteria-experiment-guide/
• ↑ https://www.sciencecompany.com/Bacteria-Growing-Experiments-in-Petri-Plates.aspx
• ↑ https://www.giftofcuriosity.com/growing-bacteria-in-a-petri-dish-stem-activity-for-kids/
• ↑ https://sciencing.com/sterilize-petri-dishes-5892646.html
• ↑ https://www.addgene.org/protocols/inoculate-bacterial-culture/

About This Article

If you want to grow bacteria in a petri dish, prepare an agar by mixing 1/2 teaspoon of agar powder with 1/4 cup of water. Place the mixture in the microwave for 1 minute until the water boils, and then let it cool to room temperature before pouring the mixture into the dish. Move the dish to the refrigerator until you need them, and then introduce the bacteria by rubbing a swab or dropping liquid onto the agar. Then, use a piece of tape to label and seal the dish, and place it in a dark, warm place for 4-6 days to grow the bacteria. For tips on recording your results and getting rid of the dishes, read on! Did this summary help you? Yes No

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Although we often think about bacteria as something we need “wash off” so we don’t get sick, the truth is that bacteria is EVERYWHERE.

And when I say that bacteria is everywhere, I mean that we are literally covered in bacteria both inside and out. In fact, we couldn’t live without bacteria as bacteria are instrumental in so many functions. For example, bacteria break down carbohydrates in our guts and prime our immune systems to fight infection.

But how do you teach kids about bacteria when it can’t be seen with the naked eye? And how do you show them that bacteria is literally everywhere around us?

Well, I enlisted the help of my mother (a retired, award winning science teacher) to help us culture and grow some bacteria. Growing bacteria in a petri dish was a very impactful activity for both of my children, making visible to them that which is usually invisible.

Note: Find more science experiments on my Science Activities for Kids page!

We started by talking about how bacteria are single-celled organisms that are neither animals nor plants. They are so tiny that we can’t see a single bacteria with our naked eye, but we can see bacteria when it grows in clump-like groups.

We discussed how there is bacteria ALL OVER. We brainstormed some places we might find bacteria, such as door handles, keyboards, dirt, etc.

Then we told the kids were were going to grow some bacteria so we could see it. We were also going to discover what things carry more bacteria and what things carry less.

My mom showed the kids how to swab items around the house for bacteria. At first, for training purposes, we used q-tips to swab things like our keyboard.

We also swabbed our necks.

Then my mom showed them how they would lightly run the swab over the agar to culture it with bacteria. (During this practice phase they simply ran their q-tips over the lid of our petri dish rather than the agar itself. In this way the agar remained sterile.)

Once we were done learning how to swab for bacteria, we talked a bit about our petri dishes. A petri dish is a round, shallow, clear-colored dish with a lid used to culture microorganisms such as bacteria. I purchased a set of petri dishes  that were pre-filled with agar, a substance that bacteria like to feed on.

We labeled the bottom of our petri dish with the items we planned to culture. In the image below, you can see that we labeled this petri dish to grow bacteria cultured from our TV remote and the bottom of my son’s foot.

(Be sure to label the bottom of the petri dish rather than the lid, as the lid will turn, causing your label to be over the wrong part of your dish.)

Next, we opened the sterile swabs that came with our set of petri dishes .

Now that the kids were experts in swabbing items, they used the sterile swabs to gather bacteria from several objects. In the photo below, my daughter was collecting bacteria from Baby, her stuffed monkey.

The kids then gently rubbed the swab over the agar in the part of the petri dish labeled for each object. It is crucial that you rub gently here so as to transfer bacteria to the agar without poking holes in the agar.

After we cultured our petri dishes with swabs from several household items, we set the dishes under a desk lamp to keep the temperature around 95 degrees F / 35 degrees C.

If the temperature is too low, bacteria will still grow, but the process will be very slow.

If the temperature is too hot (over 100 degrees F / 37 degrees C), some types of bacteria will not be able to survive.

We used a strip thermometer (similar to, but not identical to, this one from Amazon) so we could ensure the temperature was just right.

By the second day, we could see some bacteria growing in our petri dishes. And by day 6 (pictured below), we had a LOT of bacteria.

These photos clearly show growth of multiple different strains of bacteria (as evidenced by the different colors and textures).

Of the four items we swabbed for our bacteria cultures, my daughter’s stuffed monkey, Baby, was the clear winner in terms of how much bacteria we were able to grow from that swab.

We have all had a good laugh over the fact that her stuffed monkey – that she hugs and kisses and sleeps with every night – has more bacteria than the bottom of her brother’s foot. 🙂

More science activities for kids

More science posts from Gift of Curiosity:

• Dancing raisins
• Candy experiments
• Jumping colors science activity
• Make your own glycerin soap
• Crystallized snowflakes
• Dissecting an apple
• What do ants like to eat?
• Make your own telescope
• The great baking soda and vinegar experiment
• Magic inflating balloons

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Grow Bacteria from Items in Your Home

(Biology for ages 8+)

Do you wash your hands after using the bathroom or before eating? Have you ever helped your parents clean the bathroom or the kitchen? I bet you’ve noticed that all of these things tend to get dirty again  and you just have to clean them again and again! What’s the point of cleaning anyways?

Just because something looks  clean doesn’t mean it is actually  clean. There are teeny, tiny creatures that live everywhere in the world, including on your skin, on your television, and even inside your body!

These creatures are called bacteria and they are microorganisms, meaning you need a microscope to see them because they are too small to be seen with the naked eye. Most bacteria in the world are harmless, and some are even helpful, like the ones that live inside your gut to help you digest the food you eat.

But there are also bacteria that cause diseases that can be very harmful and even deadly sometimes. They cause diseases like food poisoning (or the stomach flu), strep throat, and pneumonia.

Cleaning is important because you get rid of the bacteria that are living around your house and on your body. Even though most of these won’t hurt you and some even protect you from the harmful bacteria, it is important to clean things every now and then to keep yourself from running into any bad ones that may have snuck in.

The video above shows a really cool way of comparing the bacteria that are present before and after cleaning to show how important it is to practice good hygiene. Here’s what you’ll need:

EITHER sterilized Petri dishes and nutrient agar powder

OR pre-poured agar Petri dishes

Cotton swabs

Cup of clean, room-temperature water (distilled if possible)

Clorox wipes or other household disinfectant cleaner

Zip-lock bags

Latex gloves (optional but highly recommended)

Fine-point felt pen or marker (fine-point Sharpies work well)

Adult supervision (Adult Supervision at all times please)

• The video above shows how to make your own agar and prepare your petri dishes at home. You can easily purchase nutrient agar and sterilized Petri dishes online and closely follow the directions to prepare the agar. You can also purchase Petri dishes that have been prepared with agar.
• The agar-prepared Petri dishes will be used to grow colonies of bacteria that you collect from surfaces and objects around your home both before and after the surface or object has been cleaned. Keep track of your Petri dishes by labeling them with the fine-point marker or setting them on a labeled paper or card that designates the surface you swabbed it with.

**Pro-tip: Make the most of your Petri dishes by using one dish for both the before and after swabs of the same surface. Using the fine-point felt marker, draw a straight line down the middle of the bottom half of the Petri dish (the half with the agar in it). Do not write on the agar, but the plastic dish itself.

Then label the side that will be used for the before swab by writing a small “B” along the edge, and writing a small “A” along the edge to mark the after side. It is important to make small marks along the edges because large writing in the middle will make it more difficult to see the bacterial colonies that grow.

• Dip a clean cotton swab into your cup of water to moisten it, and then wipe it over your test surface. Immediately wipe this swab on the agar of the appropriately labeled Petri dish. If you followed the pro-tip advice above, be sure to only wipe the swab on the correct side of the Petri dish and do not cross the middle line.
• Next, clean the surface or object you just swabbed. Use Clorox wipes or another household cleaner to thoroughly clean it and then let it dry.
• Once the test surface is dry, using a new cotton swab, again dip the swab into the cup of water and then wipe the cleaned surface. Immediate wipe this swab on the agar of a new Petri dish or the “after” side if you followed the pro-tip advice.
• Repeat steps 2 through 6 for each surface or object you would like to test. We recommend things that are often touched, like door knobs, light switches, your favorite toy, the toilet seat, the toilet handle, phone, television remote or game console controllers.
• Once you have swabbed all of your Petri dishes, place them flat in a box or other darkened space to allow the bacteria to grow undisturbed. For best results, wait one week before rechecking the Petri dishes. Don’t forget about them though! Set an alarm or reminder to check them so the bacterial colonies do not grow out of control.
• When it comes time to check on your bacteria, we recommend wearing latex gloves when touching them to avoid picking up any bacteria that may be harmful. Talk about what you see. Are there big differences between the before and after bacteria? Do you see some surfaces or objects that grew lots of bacteria compared to others? Do the bacterial colonies all look the same or do you see different shapes and sizes?
• After you are finished checking out your bacteria, be sure to safely dispose of the Petri dishes by sealing them in a zip-loc bag before throwing them in the garbage. Remember to properly wash your hands after handling the bacterial colonies.

The agar in the Petri dishes is a specially designed “jelly” that has tons of nutrients and sugars to provide nourish the bacteria you collect. You may never see the fuzzy bacterial colonies on the surfaces of your phone or toilet handle like you see after a week on the agar.

That’s because there usually are not a ton of nutrients present on those surfaces in your house so the bacteria do not grow as much. They still exist on those surfaces and when you place them on the agar, they are able to grow very quickly so we can see them on the agar jelly.

After you clean the surfaces and objects in your house, most of the bacteria living on them are gone so there are likely fewer and smaller colonies from the cleaned surfaces.

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• Bacteria Growing

Bacteria Growing Experiments in Petri Plates

Introduction.

Bacteria are microorganisms that grow everywhere. We can collect and grow them in specially prepared petri dishes. Nutrient agar is an excellent medium for supplying bacteria with nutrients and an environment in which we can see them grow.

Sterile powdered agar with nutrients can be mixed with water, heated and then poured into empty petri plates or ready-to-use dishes can be purchased. The undigestible agar is a gelatin-like substance with a semi solid surface on which the bacteria can grow while they consume the added nutrients. Therefore gelatin itself does not make a good growing medium. Some bacteria can digest gelatin, which is a protein derived from animal tissue. This destroys the growing surface in the petri plate making it unsuitable as a bacteria growth medium.

CAUTION. Most bacteria collected in the environment will not be harmful. However, once they multiply into millions of colonies in a petri dish they become more of a hazard. Be sure to protect open cuts with rubber gloves and never ingest or breathe in growing bacteria. Keep growing petri dishes taped closed until your experiment is done. Then you should safely destroy the fuzzy bacteria colonies using bleach.

Below are general outlines of three types of experiments involving bacteria growth. They are offered to assist in designing your own experiment or project.

Experiment 1: Direct Contact

Discussion..

In this type of experiment, bacteria is transferred directly to the prepared petri plate via direct contact. You can test the effectiveness of different soaps by treating different petri dishes with "dirty" hands before washing and "clean" hands after washing. Or, you can press a variety of common objects like coins, combs, etc. on different plates and compare the bacteria growth that results.

What you need.

• Prepared petri plates containing agar medium and nutrients.
• Bacteria on hands, paws, etc.
• Wax pencil for labeling dishes.
• Masking tape.

What to do.

• Prepared petri dishes should be refrigerated until used and always stored upside down (i.e media in upper dish, cover on bottom). This keeps condensation which forms in the lid from dropping onto and disrupting the bacteria growing surface.
• When ready to use, let dishes come to room temperature before taking samples (about one hour).
• Without tearing the agar surface, inoculate the dish by gently pressing fingers, finger nails, coin, etc onto agar surface. (Direct contact of lips or tongue is NOT a good idea.)
• Replace cover on dish, tape closed, and label each dish so you know the source of the bacteria. Store upside down.
• Let grow in undisturbed warm location. Bacteria can grow at any temperature from about ambient room temperature (hopefully around 70°F) all the way up to about 100°F. Do not place in sunlight or on a heating register.
• You should see growth within a couple of days. The dishes will start to smell which means the bacteria are growing.
• Make observations and keep records of what you see growing in each dish. Can you make any conclusions about what objects had the most bacteria?
• Before disposing of dishes in the trash the bacteria should be destroyed. Pour a small amount of household bleach over the colonies while holding dish over sink. Caution - do not allow bleach to touch your skin, eyes or clothes. It will burn!

Experiment 2: Collected bacteria samples

Use a sterilized inoculating loop or sterile swabs to collect bacteria from different locations and then streak each petri dish with your sample. This involves a bit more technique than Experiment 1 but offers a wider choice of bacteria sampling locations. Swabs can be run over doorknobs, bathroom fixtures, animal mouths, etc.

• Prepared petri dishes containing agar medium and nutrients.
• Bacteria collected from doorknobs, bathroom fixtures, etc.
• Sterile swabs or inoculating loop .
• Alcohol burner (source of flame to sterilize inoculating loop).
• Collect bacteria from each location using one swab (or resterilized innoculating loop) for each new spot.
• Inoculate each dish by streaking a pattern gently across the entire agar surface without tearing into it. Another common technique is to divide each plate into four quadrants by marking the lid with a cross. Streak your sample in straight lines starting in quadrant 1. Generally, after a few days, quadrant one will show the most growth. Depending on bacteria abundance on the swab, quadrant 4 may show no grow or only a few colonies. It is sometimes easier to distinguish different bacteria types in this low growth, less cluttered area.
• Let grow in undisturbed warm location, ideally in an environment around 100° F (37° C) - not in sunlight or on a heating register.
• Make observations and keep records of what you see growing in each dish. Can you make any conclusions about what locations had the most bacteria?

Experiment 3: Testing the effectiveness of bacteria killing agents

• Antibacterial agent (soaps, disinfectants, etc.).
• Sterile water.
• Test tubes , 12 x 75mm.
• Filter paper or paper towel.
• Small containers in which to soak paper disks.
• Hole punch.
• Prepare sterilized water by boiling water and letting cool to room temperature.
• When ready to use, let petri dishes come to room temperature before taking samples (about one hour).
• Prepare antiseptic disks by using a hole punch to create paper disks out of a piece of filter paper or paper towel. Soak one disk in each antibacterial agent to be tested. Set aside until step 6.
• Collect bacteria from each location using one swab for each new spot.
• Fill a small test tube partly full of sterilized water. Dip bacteria laden swab into water. This will transfer some of the bacteria you collected into the water. Now, inoculate a petri dish by pouring the water into the dish so the entire surface is covered. Pour out excess water. Repeat for each bacteria sample using fresh water and clean test tube each time.
• Place a pretreated antiseptic disk in each inoculated petri dish.
• Replace cover on dish, tape closed, store upside down. Be sure to label each petri dish with a name or number.
• You should see growth within a couple of days. You should also see a "halo" around each disk indicating a no growth zone. Measure and compare the size of the kill zone to determine effectiveness of each antibacterial agent.

There are many variations of the basic steps outlined above. Let us know what you tested and how your experiment turned out. We'd be delighted to hear from you! Click here and use our Contact Us form.

A Shocking Bacteria Science Experiment: Grow a Germ Farm

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I used to think that my house was clean . It certainly appears clean and tidy.  There are no piles of trash or sticky countertops.   Who knew there was so much bacteria lurking? I’m so glad we did this bacteria science experiment!

We recently studied bacteria in our science program , N OEO Biology 2 , by reading a bit of  The Complete Book of the Microscope .  I love the Usborne science books because they include suggested experiments.  The book suggested taking a bacteria culture from hands, but we decided to take this bacteria science experiment a little further and culture other surfaces around our home as well to create our own little germ farm.

The results were quite shocking.

Bacteria Science Experiment

How to grow your own germ farm.

Growing bacteria cultures requires a few things:

• Agar plates
• Sterile cotton swabs
• Bottled water
• A heat source

• BIOTECH QUALITY, FRESH AND READY-TO-USE: Factory-direct fresh 10 100-mm Pre-poured sterile LB-agar plates and 10 sterile 6-inch long swabs (in 5 pouches)….
• The Safest Agar Plates On Amazon: Luria Broth(LB) agar plates are used to detect environmental microorganisms all over the world. They are the safest agar…
• Free Science Fair Project E-Book: Packed with easy-to-do science fair experiments with clear step-by-step instructions to help you win a science fair…

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• Label the cover of the agar plates with the surface you intend to swab.
• Unwrap a sterile cotton swab and pour on a little bottled water.
• Swab the surface you want to test.
• Rub the cotton swab on the agar plate.
• Place the labeled lid on top and tape it shut.
• Turn the sealed agar plate upside down so that the name is on the bottom. This lets you observe the bacteria’s growth without the label being in the way.
• Repeat as many times as you would like, using a new agar plate and a clean cotton swab with each surface.
• Place the bacteria cultures in a warm place.  Ideally, the temperature should be kept between 85 and 100 degrees.  We placed our tray of cultures in front of a space heater in the guest room.  The room stayed pretty toasty with the door shut.  I always turned the space heater off when we went to bed, for safety reasons, but the bacteria grew even with the cooler overnight temperature.

We chose surfaces that we thought might harbor bacteria, even though they looked clean.

• Dirty hands
• Clean hands (we expected this to be bacteria-free)
• Refrigerator handle
• Door handle
• Toilet seat
• Kitchen faucet
• Light switch

We looked at our germ farm each day, but let the bacteria science experiment grow for 3 full days before recording our results.

Bacteria Science Experiment Results

After 3 full days, we observed the different cultures.  I couldn’t find a way to determine exactly which bacteria, mold, and yeasts we were growing without a high-powered microscope . We were able to use our microscope that connects to our TV to notice edge lines of the cultures, but nothing really detailed.  That’s okay though.   There was still plenty to observe.

We noticed the shape, size, and color of all the growth.  The colors ranged from a cloudy white to a creamy yellow.  There were even a few spots of fuzzy green.

What surprised us was the amount of microbes growing.  My house is generally clean!  Who knew we were surrounded by so much bacteria?

In order from least germs to the most –

• Clean hands (!)
• Fridge handle

For the most part, the bacteria amounts were a little unsettling, but likely are not harmful.  There is no way for us to know if the bacteria grown is good bacteria or bad bacteria.  We are rarely sick so I’m not too worried.   Just a bit grossed out .   I have always heard that being too clean is worse for immune systems than living around a little dirt.  I guess that must be true.

We did take this as an opportunity to review proper hand-washing techniques again though.  A friend, who is a nurse, suggested singing “Happy Birthday” twice as a way to not skimp on hand washing time.

Hannah and Ben drew a few of the bacteria cultures in their science notebooks and wrote a few sentences about the experiment and what they learned.

Then we sealed the cultures up in a Ziploc bag and threw them away. They were beginning to smell a bit, which I took as a bad sign.  My husband joked that one day soon the bacteria would take over the house.

All in all, this was a fascinating experiment and one that we hope to recreate again soon.  I would love to test surfaces out in public.  Although, I’m a little afraid to see what is lurking on a Target shopping cart handle.

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Welcome! My name is Jennifer. I am a teacher at heart. Before my children were born I was a public school teacher. Now, I am a homeschooling mom of two.

Hello! Appreciate your post! My 3rd grader is attempting this project for her science fair this weekend. We did the swabs Monday. And have kept then under a lamp for the last three days but nothing has happened …..did yours get condensation?! It’s the only thing I see. 🙁 Hope we didn’t botch her project. Thank you!

Hi! We did have some condensation. The condensation formed under the lid and the bacteria grew on the agar plate. I wonder if maybe the lamp isn’t producing enough heat for the samples. Hopefully something starts to grow soon!

I’m worried about leaving a heat source on all day when we’re at work. We’re going to attempt this for the science fair. Was thinking of an aquarium with maybe two green house lights to heat, but still worried about leaving that on. Any other suggestions or thoughts???

I checked with a scientist friend and she suggested putting the bacteria samples on top of the refrigerator.

We did this with dirty hands in the classroom. I took pictures of what grew and sent them to an expert. He first reminded me not to remove the lids under any circumstances. He then told me some of what was on there. The children in my class had nasty hands. There was staphylococcus, Ecoli, and psudamonis, Just to name a couple.

Sending a photo to an expert was a great idea! It really is shocking to see what kind of germs are all around us.

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Steve Spangler's Growing Bacteria Science Experiment Kit

Purchase options and add-ons.

• POPULAR FOR SCIENCE FAIRS: Steve Spangler's Growing Bacteria Kit is our most popular kit for Science Fairs! The project is to be simple enough for young scientists to grasp, but the experiment also has the ability to be taken further!
• BECOME A MICROBIOLOGIST: Steve Spangler's Growing Bacteria Kit includes easy-to-make nutrient agar, just like microbiologists use to grow funky stuff in their lab!
• TEACH THE SCIENTIFIC METHOD: Steve Spangler's Growing Bacteria Kit includes a step-by-step scientific method guide to guide you through your experiment!
• LEARN ABOUT GERMS: This Growing Bacteria Science Experiment Kit helps students to learn about germs and where they can be found!
• ACTIVITY GUIDE INCLUDED: Everything you need for the perfect science experiment is included in the kit, all there's left to do is design your experiment, that's where our activity guide can help!

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Product details

• Is Discontinued By Manufacturer ‏ : ‎ No
• Product Dimensions ‏ : ‎ 1 x 1 x 1 inches; 1.04 Pounds
• Manufacturer recommended age ‏ : ‎ 5 years and up
• Item model number ‏ : ‎ WAGR-600
• Date First Available ‏ : ‎ May 30, 2017
• Manufacturer ‏ : ‎ Steve Spangler Science
• ASIN ‏ : ‎ B0712524PN
• #2,329 in Educational Science Kits

Product Description

Our most popular all-inclusive Science Fair Experiment using easy-to-make nutrient agar. You can smell a good science project a block away... especially when it's SICK Science. Creating an all-inclusive science fair kit can be difficult. The project has to be simple enough for young scientists to grasp, but the experiment also needs the ability to be taken further. That's what we've accomplished with the Bacteria Growing Kit. It's now wonder this kit is hugely popular with science savvy parents everywhere! The Bacteria Growing Kit begins with the easy-to-make nutrient agar. This is the same nutrient agar used by microbiologists to grow really funky stuff in the lab. From there, everything you need is right in the kit: cotton swabs, petri dishes, and a microwave-safe plastic beaker. All that's left to do is design your experiment. Don't worry, we'll help you out there, too.

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Growing Bacteria Kit

• Our most popular kit for the Science Fair
• Includes easy-to-make nutrient agar
• Step-by-step scientific method Activity Guide

You Can Smell a Good Science Fair Project a Block Away…

Learn about germs and where they can be found.

Creating an all-inclusive science fair kit can be difficult. The project has to be simple enough for young scientists to grasp, yet the experiment needs to include the ability to be taken further. It also must be creative, interesting and age-appropriate for your child. We’ve taken all of these factors into consideration with the development of our Bacteria Growing Kit. It’s no wonder this kit is hugely popular with science-savvy parents everywhere!

It Begins With the Easy-to-Make Nutrient Agar

This is the same nutrient agar that is used by microbiologists to grow really funky stuff in the lab. Everything you need to prepare and store the bacteria comes included in our Bacteria Growing Kit: cotton swabs, petri dishes — even a microwave-safe plastic beaker. All that’s left to do is design your experiment. (Don’t worry, we’ll help you out there, too!)

What Does the Bacteria Growing Kit Teach?

Our Bacteria Growing Kit experiment allows young students to observe bacteria as it grows in a petri dish. This science experiment also offers a way for older students to explore bacteria growth — what it looks like, its growth duration and how it develops over time. This experiment also helps young scientists develop an understanding of the scientific method while learning some awesome science.

What’s Included?

• Bacteria Growing Kit
• 6 large, 10 cm (3.5″) diameter Petri Dishes
• 6 cotton swabs
• 5 g (0.17 oz) of Nutrient Agar
• 600 mL Plastic Beaker
• Activity Guide

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Steve Spangler Science makes a spectacle of science! Gather 'round for hands-on science experiments that awe and inspire while teaching children how to think like scientists. With simple instructions, we make it easy to create spectacular and memorable shared learning experiences together.

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Microbial Odor Profile of Polyester and Cotton Clothes after a Fitness Session

Chris callewaert, evelyn de maeseneire, frederiek-maarten kerckhof, arne verliefde, tom van de wiele.

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Address correspondence to Nico Boon, [email protected] .

Corresponding author.

Received 2014 May 29; Accepted 2014 Aug 11.

Clothing textiles protect our human body against external factors. These textiles are not sterile and can harbor high bacterial counts as sweat and bacteria are transmitted from the skin. We investigated the microbial growth and odor development in cotton and synthetic clothing fabrics. T-shirts were collected from 26 healthy individuals after an intensive bicycle spinning session and incubated for 28 h before analysis. A trained odor panel determined significant differences between polyester versus cotton fabrics for the hedonic value, the intensity, and five qualitative odor characteristics. The polyester T-shirts smelled significantly less pleasant and more intense, compared to the cotton T-shirts. A dissimilar bacterial growth was found in cotton versus synthetic clothing textiles. Micrococci were isolated in almost all synthetic shirts and were detected almost solely on synthetic shirts by means of denaturing gradient gel electrophoresis fingerprinting. A selective enrichment of micrococci in an in vitro growth experiment confirmed the presence of these species on polyester. Staphylococci were abundant on both cotton and synthetic fabrics. Corynebacteria were not enriched on any textile type. This research found that the composition of clothing fibers promotes differential growth of textile microbes and, as such, determines possible malodor generation.

INTRODUCTION

Clothing textiles are in close contact with the microorganisms of the skin and those of the environment. The clothes create a warm and often moist environment on the skin, which leads to the growth of bacteria. In some cases, these microorganisms lead to unpleasant odors, staining, fabric deterioration, and even physical irritation, such as skin allergies and skin infections ( 1 ). The skin consists of various niches, each with its specific bacterial community present ( 2 , 3 ). Very dry areas, such as the forearm, trunk, and legs, harbor only 10 2 bacteria per cm 2 , while the axillae, umbilicus, and toe web spaces contain up to 10 7 bacteria per cm 2 ( 4 ). The human skin contains up to 19 different phyla ( 5 ) and even in one niche, the axillae, up to 9 different phyla are present ( 6 ). Skin microorganisms transfer to the clothing fibers and interact with these in several phases: adherence, growth, and damage to the fibers. Growth of bacteria is due to sweat secretions, skin desquamation, natural particles present in the clothing fibers or on the fibers itself, or nutrition from elsewhere in the environment. An important factor determining bacterium-fiber interaction is the origin and the composition of the clothing textile. A large discrepancy exists in the way bacteria adhere to natural versus synthetic fibers. It is posed that natural fibers are more easily affected by the microbiota due to the natural nutrients present in the clothing and the ability to adsorb sweat components ( 1 ). Cellulose fibers are degraded by a range of bacteria and fungi, possessing cellulolytic enzymes ( 7 ). Synthetic fibers gather moisture in the free space between the fibers but do not adsorb it on the fibers themselves. Synthetic fibers are therefore less susceptible toward bacterial breakdown, also due to the polyethylene terephthalate (PET) basis of the fiber ( 1 ).

Axillary malodor does not only emanate from the axillary skin but also from the textiles near the axillary region ( 8 , 9 ). Dravniek et al. ( 9 ) refers to this as the primary odor, originating from the axilla itself, and the secondary odor, originating from clothing in contact with the axilla. The odor would then differ between the two sites ( 10 ). It is found that a stronger body odor is generated by wearing synthetic clothing textiles compared to natural textiles ( 10 ). This is held as a common belief; nevertheless, very few published data support this finding. Much research has nonetheless been conducted on controlling body odor by adding antimicrobials to textile fabrics ( 11 – 14 ).

Corynebacterium spp. are determined as the odor causing microorganisms in the human axilla ( 15 ). It is yet unclear which microorganisms are associated with the odor formation in clothing textiles. Few studies have been performed on determining the microbiota living in clothes. Therefore, this research focuses on (i) the determination of the microbial communities living in clothes, (ii) determining whether different textiles host different communities, and (iii) determining the odor profile of different used fabrics after a sport session. This study focuses primarily on cotton (natural, consisting mainly of cellulose) versus polyester (synthetic) clothing textiles. An in vivo case study is performed on 26 healthy people, wearing 100% cotton, 100% polyester, and intermediate cotton/synthetic clothing, doing a bicycle spinning session for 1 h. A period of 28 h was left between fitness and odor assessment, in order to let the bacteria grow on the textiles. A selected and trained odor panel assessed the odor of the individual T-shirts. The bacterial community is analyzed by means of denaturing gradient gel electrophoresis (DGGE). An in vitro growth experiment is performed to analyze the selective enrichment of isolates on different clothing fabrics.

MATERIALS AND METHODS

Study design..

First, an in vivo experiment was conducted with 26 healthy subjects, wearing cotton, synthetic, and mixed cotton-synthetic T-shirts, participating in an intensive bicycle spinning session of 1 h. The T-shirts were collected, sealed in plastic bags, and stored at room temperature in the dark, so bacterial growth occurred. Axillary swabs were taken to analyze the bacterial community on the skin. Odor assessment by a trained odor panel and subsequent bacterial extraction was performed on the whole T-shirt. The individual samples were plated to obtain pure colonies for sequencing. The DNA was extracted from axillary and T-shirt samples and the microbial community was investigated by means of DGGE. Descriptive diversity and dynamics analysis was performed on the results. Second, an in vitro growth experiment was conducted in which typical skin/textile microbial isolates were incubated on a range of sterile textile fibers in order to identify the selective growth or inhibition on the textiles. Third, contact angle measurements were performed to detect the affinity of micrococci toward polyester and cotton textiles.

Samples were taken from the T-shirt and the armpit skin of 26 healthy subjects (13 males and 13 females), participating in an intensive bicycle spinning session of 1 h. The median age was 39 years old (range, 20 to 60 years old) ( Table 1 ). Every subject wore a freshly washed T-shirt. All were in good health and had not received any antibiotics for at least 2 months. The participants had no history of dermatological disorders or other chronic medical disorders and had no current skin infections. No attempts were made to control the subjects' diet or hygiene habits. All participants were residents living in the area of Willebroek (Belgium), with a temperate maritime climate by the North Sea and Atlantic Ocean. After 1 h of intensive bicycle spinning, the T-shirts were aseptically collected and separately sealed in plastic bags. The bags were kept at room temperature (20°C) in the dark for 28 h. This was done to simulate the home conditions and to let the microbial community grow on the specific clothing textiles. An axillary swab was taken from each participant, using a sterile cotton swab (Biolab, Belgium) that was formerly moistened with sterile physiological water. The swab was thoroughly swabbed for 15 s in the axillary region to detach and absorb the microorganisms, after which the tip was broken in a sterilized reaction tube filled with 1.0 ml of sterile physiological water ( 16 ). The bacterial samples were pelletized and frozen at −20°C until DNA extraction.

Metadata of the participating subjects

deo, deodorant or antiperspirant applications.

Odor assessment.

Individual T-shirts in the plastic bags were presented to a panel of seven selected and screened human assessors. Assessors were selected by means of sensitivity to dilutions of n -butanol and wastewater and by means of the triangle test ( 17 ). Each member of the panel was presented three flasks, two of which were the same while the third contained a different odor. The flask was shaken, the stopper was removed, after which the vapors were sniffed. The panelists had to correctly identify the different flask. The triangle test was repeated three times, with a minimum of 2 days in between each measurement. The room in which the tests were conducted was free from extraneous odor stimuli, e.g., such as odors caused by smoking, eating, soaps, perfume, etc. A representative team of odor assessors was chosen from the pool of assessors. The odor assessors were familiar with the olfactometric procedures and met the following conditions: (i) older than 16 years and willing to follow the instructions; (ii) no smoking, eating, drinking (except water), or using chewing gum or sweets for 30 min before olfactometric measurement; (iii) free from colds, allergies, or other infections; (iv) no interference by perfumes, deodorants, body lotions, cosmetics, or personal body odor; and (v) no communication during odor assessment. The samples were assessed by seven odor characteristics: hedonic value (between −4 and +4), intensity (scale 0 to 6), musty (scale 0 to 10), ammonia (scale 0 to 10), “strongness” (scale 0 to 10), sweatiness (scale 0 to 10), and sourness (scale 0 to 10). A control odor measurement, a clean cotton T-shirt with random number, was served to the odor panel together with the other samples.

Statistical analysis odor characteristics.

The generated data set from the odor assessment was statistically analyzed and visualized in R ( 18 ). A heat map and scatterplot were generated to visually interpret the correlations between sensory variables. Significance cutoff values were set at 95% (α = 0.05), unless otherwise mentioned in the manuscript. Both a multivariate comparison of means as well as univariate analysis were run after assessment of the hypothesis. Univariate normality was assessed using a Shapiro-Wilk normality test. If normality could not be assumed, the Mann-Whitney (or Wilcoxon rank sum) test was executed to assess null hypothesis of a location shift μ = 0. The alternative hypotheses were selected based upon exploratory data analysis. Nonavailable observations were handled by case-wise deletions. Multivariate data sets were analyzed on their normal distribution using Mahalanobis distances in quantile-quantile (QQ) plots. Also, an E-statistic test of multivariate normality was executed ( 19 ). Multivariate homogeneity of group dispersions (variances) was assessed using the betadisper function from the package Vegan ( 20 ), an implementation of the PERMDISP2 procedure ( 21 ). Euclidean distance measures were used, as well as the spatial median for the group centroid. A Hotelling's T 2 test was used to compare the multivariate data sets, comparing the multivariate means of each population ( 22 ). When necessary a chi-squared approximation was used for the test to allow for relaxation of the normality assumption.

Bacterial extraction from T-shirts.

The bacterial extraction occurred on the complete T-shirt, using TNE buffer (10 mM Tris-HCl [pH 8.0], 10 mM NaCl, 10 mM EDTA) ( 23 ). A 300-ml portion of TNE buffer was added to the plastic bag with the T-shirt, firmly sealed with tape, and vortexed for 10 min. The buffer was subsequently manually pressed out of the T-shirt and transferred into sterile 50-ml reaction tubes. The extracts were respectively used for isolation of bacteria and for DNA extraction. The bacterial extraction procedure was chosen after an optimization procedure (see Fig. S1 in the supplemental material). The method focused on the extraction of the bacteria of the whole T-shirt. It was not possible to extract the bacteria from one region (e.g., axillary region) of the T-shirt. A clean T-shirt was extracted, together with the other samples, as a control measurement.

Sanger sequencing of bacterial isolates.

The microorganisms were isolated from the T-shirts by the standard method of dilution plating on nutrient agar. Incubation of all plates was performed at 37°C in aerobic conditions and facultative anaerobic conditions using a gas-pack cultivation jar. The colonies were plated three times on new agar plates using the streak plate method to obtain bacterial isolates. A total of 91 isolates was obtained. The isolates were transferred into a 1.5-ml Eppendorf with 50 μl of sterile PCR water, vortexed, and stored at −20°C to extract DNA. Dereplication was done using DGGE after amplification by PCR using the 338F and 518R primers ( 24 , 25 ). The analysis involved 31 nucleotide sequences. The 16S rRNA genes were subsequently amplified by PCR using 63F and 1378R ( 26 ). The PCR program were performed and checked as described below. Sanger sequencing was performed on the 16S rRNA amplicons, aligned, and compared to sequences from the National Center for Biotechnology Information (NCBI) database. The closest match of each isolate was identified. The bacterial isolates were constructed in an evolutionary taxonomic circular tree (see Fig. 2 ) using the neighbor-joining method ( 27 ), conducted in MEGA5 ( 28 ). The tree has branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Jukes-Cantor method ( 29 ) and are in the units of the number of base substitutions per site. The codon positions included were first + second + third + noncoding. All ambiguous positions were removed for each sequence pair. There were a total of 1,172 positions in the final data set.

Bacterial isolates obtained from the T-shirts after the spinning session represented in an evolutionary taxonomic circular tree, using the neighbor-joining method.

DNA extraction, PCR, and DGGE.

The bacterial solution in the TNE buffer was centrifuged for 5 min at 6,000 × g . The supernatant was discarded, and the obtained pellet was used for further DNA extraction. Total DNA extraction was performed using an UltraClean water DNA isolation kit (Mo Bio, USA). The DNA was stored at −20°C until further analysis. The DNA extraction was chosen after a comparative study of different DNA extraction methods (see Fig. S2 in the supplemental material). The 16S rRNA gene regions were amplified by PCR using 338F and 518R ( 24 , 25 ). A GC clamp of 40 bp ( 24 , 25 ) was added to the forward primer. The PCR program consisted of 10 min 95°C, followed by 35 cycles of 1 min at 94°C, 1 min at 53°C, and 2 min at 72°C, with a final elongation for 10 min at 72°C. Amplification products were analyzed by electrophoresis in 1.5% (wt/vol) agarose gels stained with ethidium bromide. DGGE was performed as previously reported ( 6 ). A control measurement was taken into account. To process and compare the different gels, a homemade marker of different PCR fragments was loaded onto each gel ( 6 ). Normalization and analysis of DGGE gel patterns was done with the BioNumerics software 5.10 (Applied Maths, Sint-Martens-Latem, Belgium). The different lanes were defined, the background was subtracted, differences in the intensity of the lanes were compensated for during normalization, and bands and band classes were detected.

Selective growth of bacteria on textiles.

To analyze the selective growth of pure bacterial strains on different clothing textiles, bacteria were inoculated and incubated on a sterile piece of textile in an in vitro growth experiment. A wide range of clothing textiles was screened: polyester, acryl, nylon, fleece, viscose, cotton, and wool. Five common skin bacteria were grown on the textiles: Staphylococcus epidermidis CC6 (GenBank accession no. KJ016246 ), Micrococcus luteus CC27 (GenBank accession no. KJ016267 ), Enhydrobacter aerosaccus (LMG 21877), Corynebacterium jeikeium (LMG 19049), and Propionibacterium acnes (LMG 16711). The bacteria were cultivated for 48 h in nutrient broth, washed in M9 medium and finally dissolved in fresh M9 medium. A sterile piece of textile of 25 cm 2 was inoculated with 100 μl of the bacterial culture in a petri dish. The inoculated bacteria were incubated for 3 days at 37°C. The bacteria were subsequently extracted using 10 ml of TNE buffer ( 23 ). The bacterial suspensions were measured using flow cytometry. To verify the extraction efficiency of the different clothing textiles, the bacterial strains were immediately extracted after inoculation using 10 ml of TNE buffer. All experiments were carried out in triplicate. A control measurement, where bacteria were grown without textiles, was each time taken into account and deducted from the measurements.

Flow cytometry.

Flow cytometry was used as a fast microbial measurement technique. The laser detection point of the device beams one cell at the time (λ max = 488 nm), while the forward and side light scatter are detected. The samples were diluted 100 times in filtered Evian water (Danone Group, Paris, France) and stained with 1/100 SYBR green I dye (Invitrogen), as described in previous studies ( 30 ). The DNA-dye complex absorbs blue light (λ max = 497 nm) and emits green light (λ max = 520 nm). Prior to flow cytometric analysis, the stained samples were incubated for 15 min in the dark at room temperature. Every sample was measured in triplicate, using a BD Accuri C6 flow cytometer (BD Biosciences, Belgium). The measurements were processed using the BD Accuri C6 software.

Contact angle measurements.

The affinity of micrococci ( Micrococcus luteus ) toward specific clothing textiles (cotton and polyester) was measured by means of contact angle measurements on the fabrics and the micrococci, as described earlier ( 31 ). Drops of three different solutes were applied on the tissues to determine Lifshitz-Van der Waals and electron-donor and -acceptor components of the surface tension, using the Young-Dupré equation and the extended DLVO approach ( 31 ). The solutes (Milli-Q water, diiodomethane, and glycerol) had different physicochemical properties with known physicochemical parameters. Since the textile fabrics absorbed much moisture due to the large voids between the fibers, contact angles were carried out on substitute materials: PET plastic to simulate polyester fibers, since PET is the basic substance for polyester, and cardboard (cellulose) for cotton. Micrococcus luteus was cultivated in nutrient broth for 3 days at 37°C. The bacteria were filtered on a 0.45-μm-pore-size filter until a firm layer of micrococci was obtained, on which the contact angles were measured. Drop measurements were repeated at least 10 times for each liquid, whereby the average was taken. Anomalous measurements were rejected. All contact angles were measured using contact angle equipment (Krüss DSA10 goniometer; Krüss GmbH, Hamburg, Germany) equipped with contact angle calculation software (Drop Shape Analysis; Krüss GmbH).

Ethics statement.

The study was approved by the Ghent University Ethical Committee with approval number B670201112035. All participants gave their written consent to participate in this study, as well as consent to publish these case details.

Nucleotide sequence accession numbers.

Sequences for all of the strains were submitted to GenBank under accession numbers KJ016241 to KJ016271 .

Odor differences between cotton and polyester clothing textiles.

The hedonic value (i.e., the pleasantness of the odor) was qualified by the odor panel on a scale from −4 (very unpleasant) to +4 (very pleasant). The average hedonic value of 100% cotton T-shirts was −0.61 ± 1.08, while for 100% polyester T-shirts, a significantly lower value of −2.04 ± 0.90 was determined (see Table S1 in the supplemental material). Polyester clothing after the spinning session smelled significantly less pleasant, and additionally, more intense, more musty, more ammonia, more strong, more sweaty and more sour ( Fig. 1 ). The qualitative differences were the largest for the sourness, strongness, and mustiness. The data set of the odor analysis was examined on its multivariate normal distribution by means of Mahalanobis QQ-plots (data not shown). Deviation from the bisector and, as such, from multivariate normality was observed, as confirmed formally by the E-statistic test ( P < 0.05). The multivariate means of cotton and polyester were compared to each other with the Hotelling two-sample T 2 test. This gave a P value of 5.72 × 10 −6 , meaning that a significant difference was found between the multivariate means of the cotton and polyester samples. The correlations between the different variables are visually represented in the heat map in Fig. S3 in the supplemental material. The t test indicated no differences in deodorant/antiperspirant use among the 100% cotton and 100% polyester group ( P = 0.86) ( Table 1 ).

Odor characterization of cotton (green) and polyester (red) clothing after a fitness experiment, assessed by the odor panel. The hedonic value was assessed between a value −4 (very unpleasant), 0 (neutral), and +4 (very pleasant) and rescaled between 0 and 8. The intensity represents the quantity of the odor, in a value between 0 (no odor) and 10 (very strong/intolerable). The qualitative odor characteristics musty, ammonia, strongness, sweatiness, and sourness were assessed between 0 and 10. The odor assessment is represented in box plots, with the middle black line as the median odor value and the small circles as the outliers. Polyester clothing smelled significantly more after a fitness session than cotton.

Bacterial isolation and identification.

Isolates of pure bacterial colonies were identified and are represented in Fig. 2 . A total of 91 isolates was obtained from aerobic and anaerobic plating. The isolates were screened by DGGE and sequenced to allow identification. Figure 2 represents 31 unique species found on the T-shirts. Not only Gram-positive but also many Gram-negative bacteria were found. Many skin-resident staphylococci were isolated from the textiles. Isolates also belonged to the Gram-positive Bacillus spp., Gram-positive Micrococcus spp., and Gram-negative Acinetobacter spp. and to the Gram-negative Enterobacteriaceae family, among others, which are generally not found on the axillary skin. The isolates were classified into three bacterial phyla: Firmicutes , Actinobacteria , and Proteobacteria .

Bacterial fingerprinting of the textile microbiome.

DGGE fingerprinting analyses showed large diversities among the individual shirts. Although similar bacterial species were noticed, every textile microbiome was rather unique. Figure 3 shows the fingerprinting results of the 26 individual T-shirts. Apparent differences were found between cotton and synthetic clothing textiles after the fitness session. Particular bands were identified that correlated more with specific clothing fibers. Micrococcus spp. were predominantly found in synthetic clothing fabrics. Many micrococci were found on 100% polyester clothes, but they were also on mixed synthetic textiles, such as 82% polyester plus 18% elastane. Micrococci were also found on mixed synthetic/natural textiles, such as 95% cotton + 5% elastane and 35% polyester + 34% cotton + 28% lyocell + 3% elastane ( Fig. 3 ). Staphylococcus hominis bands were solely present on the 100% cotton clothing. Staphylococcus spp. were detected in relatively large amounts in practically all T-shirts. Individual DGGE fingerprinting was performed on both textiles and axillary skin (see Fig. S4 in the supplemental material). The axillary region was chosen as a representative skin area and compared to the textile microbiome, since both are known to generate malodor. Large differences were seen in the bacterial fingerprint patterns between the axillary and textile microbiome. An enrichment of skin bacteria on the textile was frequently observed, such as the apparent enrichment of Staphylococcus epidermidis ( Fig. 3 ). The fingerprint results show that selective bacterial growth occurs in synthetic and cotton clothing.

DGGE bacterial profile of 26 individual T-shirts after the bicycle spinning session. The legend on the right represents the subject number, and the textile fibers are indicated as follows: P, polyester; C, cotton; E, elastane; and L, lyocell. The samples were separated between cotton and synthetic clothing fibers.

Selective bacterial growth on clothing textiles.

The selective growth of pure bacterial cultures was examined by means of an in vitro growth experiment on a range of different fabrics. The results, presented in Table 2 , clearly indicated selective growth and inhibition for several species on the different fabrics. Enhydrobacter aerosaccus and Propionibacterium acnes were able to grow on almost every textile. Under the same conditions, Corynebacterium jeikeium was not able to grow on the textiles, as the log counts decreased. Staphylococcus epidermidis was able to grow on almost every textile, except viscose and fleece. Propionibacterium acnes showed a remarkable growth on nylon textile, with bacterial counts up to 2.25 × 10 8 CFU per cm 2 . The log count difference among textiles was the most dissimilar for Micrococcus luteus . The largest growth was noted on polyester textiles (1-log growth increase; up to 1.72 × 10 7 CFU per cm 2 ), whereas the largest inhibition was noted on fleece textiles. This experiment confirmed the finding of selective growth of Micrococcus spp. on polyester clothing textiles, as well as no selective growth of Micrococcus spp. on cotton textiles. According to these results, viscose did not permit any growth of bacterial species. Wool, on the other hand, supported the growth of almost all bacteria. Nylon showed very selective bacterial growth. The growth of Staphylococcus , Propionibacterium , and Enhydrobacter spp. was enhanced, while the growth of Micrococcus and Corynebacterium spp. was inhibited. Growth on fleece likewise showed a selective profile. Enhydrobacter spp. were enhanced, Propionibacterium and Corynebacterium spp. remained at the same level, and Staphylococcus and Micrococcus spp. were inhibited. No growth (or inhibition) was observed on acryl textile for practically all species. Cotton textile indicated a growth for Propionibacterium , Staphylococcus , and Enhydrobacter spp., while practically no growth (or inhibition) was noted for Micrococcus and Corynebacterium spp. Polyester textile was associated the greatest growth for Propionibacterium , Enhydrobacter , and Micrococcus spp. Inhibition was recorded for Corynebacterium spp. on polyester. No growth (or inhibition) was noted for Staphylococcus spp.

Growth or inhibition (in log numbers) of bacterial species after a 3-day inoculation on different clothing textiles a

Average CFU/cm 2 of the triplicates are represented, together with the standard deviations. A color code is given according to the log growth or reduction compared to the initial bacterial concentration.

A potential explanation for the selective growth is a dissimilar nonelectrostatic attraction between the bacterium and the different textile surfaces. Contact angle measurements were carried out (see Table S2 in the supplemental material) to determine the attraction or repulsion for Micrococcus luteus toward cotton (cellulose) and polyester (PET). Using the Young-Dupré equation, the contact angles were transformed into surface tension components, represented in Table S3 in the supplemental material. The interaction energy between micrococci and cotton (Δ G = −1.22 ± 1.00 J) was in the same range as the interaction energy between micrococci and polyester (Δ G = 0.24 ± 1.00 J). Both values were determined to be around 0. No differences were found in the interaction energies for micrococci and cotton and for micrococci and polyester.

It is generally accepted that the choice of clothing has an impact on malodor formation ( 10 ). This research showed that polyester clothes create a significantly higher malodor compared to cotton clothing after a fitness session and an incubation period. Significant differences were found for the hedonic value and the intensity of the odor, as well as all qualitative odor characteristics (musty, ammonia, strongness, sweatiness, and sourness). This corroborates earlier findings, where higher odor intensities were detected in polyester fabrics ( 10 ). The first reason for the different odor profile is explained by the difference in odor adsorbance. Polyester is a petroleum-based synthetic fiber and has no natural properties. Synthetic fibers hence have a very poor adsorbing capacity, due to their molecular structure. Cotton is a natural fiber, originating from the Gossypium cotton plants. These cotton fibers almost purely consist of cellulose, which has a high adsorbing capacity ( 32 ). Next to moisture, odors are adsorbed, and less malodor is emitted. A second reason can be explained by the dissimilar bacterial growth on the different textiles, where the malodor causing Micrococcus spp. tends to grow better on synthetic textiles. The poor adsorbing properties and the selective bacterial growth of micrococci may account for the malodor emission by certain synthetic sport clothes.

The microbial community of the textiles differs with the community living on the axillary skin (see Fig. S4 in the supplemental material). While the axillary microbiome is generally dominated by Staphylococcus and Corynebacterium species ( 6 ), the textile microbiome was rather dominated by Staphylococcus and Micrococcus spp. ( Fig. 3 ). The three main bacterial phyla found in the textiles ( Firmicutes , Actinobacteria , and Proteobacteria ) are also three important phyla of the skin microbiome ( 5 ). Certain species were able to grow in more abundant quantities on the textile fibers. It is suggested that malodor generation is associated with the selective growth of those species. The bacterial enrichment was studied and differed depending on the bacterial species and the type of clothing textile, as shown by an in vitro growth experiment ( Table 2 ). Micrococci were selectively enriched on polyester and wool but were inhibited on fleece and viscose. Polyester textiles showed an enrichment for Micrococcus , for Enhydrobacter , and Propionibacterium spp. These enrichments can have an important impact on the malodor creation from excreted sweat compounds. Staphylococcus epidermidis was enriched on both cotton and polyester textiles, as seen in the fitness clothes ( Fig. 3 ). These results are in close correlation with previous findings, where a high affinity of Staphylococcus spp. for cotton and polyester was reported ( 33 , 34 ). The enrichment was confirmed by the in vitro growth experiment, with a growth reaching up to 10 7 CFU per cm 2 textile for cotton, wool, and nylon. On polyester, the presence was maintained on a level of 10 6 CFU per cm 2 . In addition, Staphylococcus hominis was often able to gain dominance on cotton textiles, as seen in the fitness experiment. This was not seen for synthetic clothing textiles. No bacterial enrichment was seen on viscose, a textile made from regenerated wood cellulose. Viscose showed very low bacterial extraction efficiencies. Further research is needed to confirm the absence of bacterial growth on viscose. If bacterial growth is indeed impeded on these fiber types, viscose could be used as bacterium- and odor-preventing textile in functional clothes. Wool, on the other hand, promoted the growth of almost all bacteria. This is in correlation with earlier findings, where the highest bacterial growth was noted for wool compared to the other tested clothing textiles. Although wool was associated with high bacterial counts, the odor intensity ratings were the lowest for wool ( 10 ). nylon showed a very selective bacterial growth, with the biggest enrichment noted for Propionibacterium spp. (up to 10 8 CFU per cm 2 ). Staphylococcus and Enhydrobacter spp. were enhanced as well, whereas the growth of Micrococcus and Corynebacterium spp. were inhibited. The Propionibacterium spp. are known to cause an acidic, intense foot odor ( 35 ). The enrichment of these species on nylon socks has an important consequence on the foot malodor generation.

The Corynebacterium genus was not able to grow under the circumstances of the in vitro growth experiment. The genus was likewise not detectable by DGGE, nor could it be isolated from any clothing textile after the fitness experiment, although it was initially present in the axillae of many subjects (see Fig. S4 in the supplemental material). These findings are consistent with previous findings, where no growth of corynebacteria on clothing textiles was found ( 10 , 34 ). Corynebacteria are generally known as the most important species causing axillary malodor ( 36 ). These bacterial species are thought to be involved in the conversion of sweat compounds into volatile short branched-chain fatty acids, steroid derivatives, and sulfanylalkanols—the three main axillary malodor classes ( 15 ). The results of the present study, together with former research, indicated that corynebacteria are not the abundant bacterial species on clothing textiles. The absence or inability of corynebacteria to grow on clothing textiles implies that there are other bacterial types involved in the malodor creation in fabrics.

This research showed an overall enrichment of micrococci on the synthetic fabrics after the fitness session and incubation period. The bands were clearly visible on DGGE, meaning that the bacteria were present for at least more than 1% of the bacterial community ( 37 ). Isolates of Micrococcus spp. were identified not only in 100% polyester textiles but also in almost every shirt where synthetic fibers were present ( Fig. 3 ). The results were confirmed by the in vitro growth experiment ( Table 2 ). Of the seven tested textile types, micrococci were able to gain the highest abundance on polyester fabrics (up to 10 7 CFU per cm 2 ). No selective growth was found for micrococci on cotton textiles after 3 days. Previous research found a single enrichment of micrococci on polyester ( 34 ). These findings confirm that micrococci are selectively enriched on polyester fabrics. It is hypothesized that the circumstances on synthetic clothing textiles are favorable for the growth and activity of Micrococcus spp. Their enrichment was not caused by a higher nonelectrostatic adsorption affinity for polyester. Other factors play a role in the enrichment of the micrococci. The aerobic growth conditions on polyester favor the growth of aerobic micrococci. Bacteria in clothing textiles are no longer suppressed by the innate immune system present on the skin. The nutritious environment, as well as quorum sensing ( 38 , 39 ), can additionally play a role in the growth of micrococci. A multiplicity of these favorable situations causes the selective enrichment of micrococci on polyester fabrics. Micrococcus spp. are known for their ability to create malodor from sweat secretions. They are able to fully catabolize saturated, monounsaturated, and methyl-branched fatty acids into malodor compounds ( 4 , 40 ). Next to corynebacteria, micrococci have been held responsible for the formation of body odor. These species have a high GC% content and are related to corynebacteria (both are members of the Actinobacteria phylum). Micrococci were frequently found in the axillary region, yet always by means of culturing techniques ( 4 , 41 ). In molecular studies, micrococci have not been found in large quantities on the human axillary skin ( 6 , 42 ). We suggest that micrococci were detected as they preferentially grow on the textiles worn close to the axillae and due to the practice of culturing techniques, which favor the growth of micrococci. It is suggested that micrococci prefer the aerobic environment of the textile fibers, whereas corynebacteria prefer the lipid-rich and more anaerobic environment on/in the (axillary) skin ( 43 ). This may also explain the odor differences frequently perceived between axillary skin and the textile worn at the axillary skin. The use of underarm cosmetics may additionally impact the skin microbiome and the subjects body odor. Stopping or resuming deodorant/antiperspirant usage leads toward an altered underarm microbiome. Especially the use of antiperspirants causes significant changes ( 44 ). Other factors include the general hygiene habits (frequency of washing, soap/shower gel type, etc.), the occupational lifestyle (physical activities, food habits, etc.), and the environment (place of residence and work, climate, humidity, etc.) which can impact the skin microbiome.

This research indicated that enrichment of micrococci occurred on polyester and, in general, on synthetic clothing textiles. Micrococci were frequently isolated, identified by means of DGGE fingerprinting, and enriched by an in vitro growth experiment on these textiles. The odor of the synthetic textiles was perceived as remarkably less pleasant after an intensive sport session. Microbial exchange occurs from skin to clothing textiles. A selective bacterial enrichment takes place, resulting in another microbiome compared to the autochthonous skin microbiome. The enrichment depended on the type of clothing textile and the type of bacterial species. With the current knowledge, the textile industry can design adjusted clothing fabrics that promote a non-odor-causing microbiome. This research opens perspectives toward better and functionalized sports clothing, which emit less malodor after use. Antimicrobial agents may be added to washing machine powders specifically against the odor causing microbiota, rather than using broad-spectrum antimicrobials. The enhancement of the non-odor-causing bacteria and the inhibition of the odor-causing bacteria, which are enriched on certain textiles, could greatly improve the quality of the fabrics.

Supplementary Material

Acknowledgments.

This research was funded by the Flemish Government and Ghent University through the assistantship of C.C. F.-M.K. was supported by a research grant from the Geconcerteerde Onderzoeksactie (GOA) of Ghent University (BOF09/GOA/005).

C.C., E.D.M., T.V.D.W., and N.B. designed the experiments. E.D.M. and C.C. performed the experiments and analyzed the data. The statistical analysis was done by F.-M.K. The contact angle measurements and analysis was made possible by A.V. C.C. wrote the paper. A.V., T.V.D.W., and N.B. commented on the manuscript.

We acknowledge the odor panel and the persons attending the spinning session for their willingness to participate in this research. We thank Tim Lacoere for his assistance during the molecular work. We thank Francis de los Reyes III and Eleni Vaiopoulou for their critical review of the manuscript and the inspiring discussions.

The authors declare that they have no conflict of interest.

Published ahead of print 15 August 2014

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.01422-14 .

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An Optimized Method for Reconstruction of Transcriptional Regulatory Networks in Bacteria Using ChIP-exo and RNA-seq Datasets

• Published: 11 November 2024

Cite this article

• Minchang Jang 1   na1 ,
• Joon Young Park 1   na1 ,
• Gayeon Lee 1 &
• Donghyuk Kim   ORCID: orcid.org/0000-0001-6489-032X 1  

Transcriptional regulatory networks (TRNs) in bacteria are crucial for elucidating the mechanisms that regulate gene expression and cellular responses to environmental stimuli. These networks delineate the interactions between transcription factors (TFs) and their target genes, thereby uncovering the regulatory processes that modulate gene expression under varying environmental conditions. Analyzing TRNs offers valuable insights into bacterial adaptation, stress responses, and metabolic optimization from an evolutionary standpoint. Additionally, understanding TRNs can drive the development of novel antimicrobial therapies and the engineering of microbial strains for biofuel and bioproduct production. This protocol integrates advanced data analysis pipelines, including ChEAP, DEOCSU, and DESeq2, to analyze omics datasets that encompass genome-wide TF binding sites and transcriptome profiles derived from ChIP-exo and RNA-seq experiments. This approach minimizes both the time required and the risk of bias, making it accessible to non-expert users. Key steps in the protocol include preprocessing and peak calling from ChIP-exo data, differential expression analysis of RNA-seq data, and motif and regulon analysis. This method offers a comprehensive and efficient framework for TRN reconstruction across various bacterial strains, enhancing both the accuracy and reliability of the analysis while providing valuable insights for basic and applied research.

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Data Availability

The reference code implemented on the Google Colab platform and the test datasets required for running the pipeline, including ChIP-exo and RNA-seq datasets, are publicly available on GitHub ( https://github.com/SBML-Kimlab/TRNanalysis ). The whole datasets of ChIP-exo and RNA-seq has been deposited to GEO with the GSE271231 and GSE271232, respectively.

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Acknowledgements

This research was supported by National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (MSIT) [2021M3A9I4024840, 2022M3A9I5018934, RS-2023-00208026, RS-2024-00398252].

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Minchang Jang and Joon Young Park contributed equally to this work.

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School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, 44919, Republic of Korea

Minchang Jang, Joon Young Park, Gayeon Lee & Donghyuk Kim

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M. Jang, J. Y. Park and D. Kim designed the study. M. Jang and J. Y. Park performed experiments and conducted computational analysis. M. Jang, J. Y. Park and D. Kim drafted the manuscript. M. Jang, J. Y. Park, G. Lee and D. Kim edited the manuscript. D. Kim provided mentorship, resources and guidance in planning and implementation. All authors participated in writing the paper.

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Correspondence to Donghyuk Kim .

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Jang, M., Park, J.Y., Lee, G. et al. An Optimized Method for Reconstruction of Transcriptional Regulatory Networks in Bacteria Using ChIP-exo and RNA-seq Datasets. J Microbiol. (2024). https://doi.org/10.1007/s12275-024-00181-6

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Received : 02 July 2024

Revised : 08 October 2024

Accepted : 09 October 2024

Published : 11 November 2024

DOI : https://doi.org/10.1007/s12275-024-00181-6

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