Bacteria cultures in petri dishes

[procrastinationking]

Okay so I thought I would check bacteria growing resistance to a certain disinfectant solution [clorox disinfectant wipes (squeezed out while wearing sterile gloves)]. So I had five dishes with bacteria cultures (the same type it, it seems), and I poured the solution into each of them. I waited 30 seconds, because it says to treat a surface leave it visibly wet for 30 seconds. I expected the cultures to disappear (like in the commercials the bacteria disappears in the dramatization) but they didn't. So my question is: If bacteria die in petri dishes, do they disappear?

I used 9cm petridishes, each with about 10 ml of agar solution. I used one clorox wipe per dish.
Thank you for your time.

[donnahardy2]

Hi,

If the bacteria were exposed to Clorox, they will be dead, but they won't disappear. The colonies will still be present on the agar surface. Remember the law of conservation of matter:

"Law of Conservation of Matter: During an ordinary chemical change, there is no detectable increase or decrease in the quantity of matter"

So the dead bacteria will still be present. The only way to tell if the bacteria are dead would be to transfer them to a new Petri dish with nutrient agar and see if they will grow up again. Injured bacteria would take longer to recover and grow, so this would take a few days to determine if they are alive or dead.

If you have time and materials available, then you should test the viability of the bacteria. If you don't, and your write up is due tomorrow, then you should do a good job explaining why your experiment didn't work, and what you would do differently next time to improve the experimental design. It's common for experiments not to work, and science fair judges and teachers will give you credit for a good explanation.

Another suggestion for your write up. You should look at the product label and use the scientific name of the chemical in the Chlorox wipes in your write up.

Good luck.

Donna Hardy

[procrastinationking]

Thank you very much for your reply. I now know what to do if I am not able to finish in time.

The only way to tell if the bacteria are dead would be to transfer them to a new Petri dish with nutrient agar and see if they will grow up again. Injured bacteria would take longer to recover and grow, so this would take a few days to determine if they are alive or dead.

When transfering bacteria from one petri dish to another, is it necessary to use a inoculating loop? Or is it okay if I use a cotton swab (Q-tip) from an unopened package? Also, to get the bacteria from the original petri dish do I scrape the surface of the agar and swab it onto the new dish?

Although you said that that was the only way, is it possible to some how measure the area of dead bacteria? I found this article (http://www.princeton.edu/pr/pwb/98/0323/0323-1a.html) the other day and it's about a girl who did an experiment very similar to mines. In it it says, "After treating bacteria-infested gel plates with various pine oil disinfectants, she measured the area of dead bacteria."

Though this may be irrelevant...After reading that article I thought people would think I copied her project, so since I did not read donnahardy2's reply, I thought the clorox did not work. I then used an antiseptic (Germ-X) and applied it to each dish. So now I am doing my project on antiseptics. Therefore my question is, when writing out my procedures, should I include the disinfectant step or can I pretend I did not applied the Clorox solution and go straight to the antiseptic part?

Thank you for any help.

[donnahardy2]

Hi,

Since you don't know the number of bacteria that were on your plate before you used the Chlorox, it will not be possible to measure the number of bacteria killed. But if you have a control plate (bacteria not exposed to antiseptic), you could measure the time it takes for control and Chlorox-treated bacteria to appear on a plate after inoculation. At least that would give you a measurement. If there is a delay in the time it takes treated bacteria to grow back, you could conclude that these bacteria were damaged by the Chlorox. If they don't grow back at all after a few days, and your control bacteria do grow, then you can conclude that the Chlorox killed them.

You can use either a flamed, and then cooled inoculating loop, or a Q-tip from an unopened package. If you have an extra agar plate, you can include a negative control (sterile Q-tip or inoculating loop) just to show that your technique did not introduce extraneous bacteria.

From the article you referenced, I can't tell what the experimental design was. It's not clear how the "area of dead bacteria" was measured. I suppose that one way to do this would be to count the bacteria on a surface, by swabbing a specific area and then transferring the swab to a standard volume of water, and then plating out dilutions of the water. Doing this before and after antiseptic treatment would give a measurement in numbers of bacteria per square centimeter of surface before and after. But it doesn't sound like you have time to do this type of experiment at this point, so you should concentrate on what you can do, and your write up.


Let us know if you have any other questions.


Donna Hardy

[procrastinationking]

Hi, thanks so much for replying again

Since you don't know the number of bacteria that were on your plate before you used the Chlorox, it will not be possible to measure the number of bacteria killed.

What if I counted the number of colonies that were on the plates before I treated them (which I forgot to mention )? Is there a way i can measuer the number of bacteria killed?

Also if I do use time as to determine whether bacteria grows, would it be best to make a line graph (series 1 being the control plate and series 2 being the treated plate) where the data is how many bacteria colonies grew in so many hours?

I suppose that one way to do this would be to count the bacteria on a surface, by swabbing a specific area and then transferring the swab to a standard volume of water, and then plating out dilutions of the water. Doing this before and after antiseptic treatment would give a measurement in numbers of bacteria per square centimeter of surface before and after. But it doesn't sound like you have time to do this type of experiment at this point, so you should concentrate on what you can do, and your write up.

This sounds like a wonderful experiment, but I'm not too sure I understand it. And, unfortunately, I believe you are right in me not having enough time.

Thank you for replying.

P.S. I'm sorry I couldn't reply right away. I was at a camp with my history class for these past two days, with no internet connection.

[donnahardy2]

Hi,

I hope you had fun at history camp!

The problem is that each colony consists of millions of bacteria. So you cannot know how many bacteria you started with unless you had counted them before you exposed them to the Chlorox. One way to do this experiment would be to start with a contaminated surface, and count the number of bacteria present per square centimeter by swabbing the surface with a sterile swab and transferring it to an agar plate, or exposing an agar plate directly to the surface. Then you would treat the surface and repeat the counting experiment exactly the same way. This would give you a before and after count, and make your results quantitative.

If your project is due within the next few days, I recommend concentrating on your write-up rather than try to repeat the experiment, even if your results are not exactly measurable. Make sure your write up is complete and thorough and includes each section. Look at your teacher's hand-out again, and make sure you have included every item on the list, including the background section, hypothesis, materials required, procedure, results, conclusion, and bibliography. In your conclusion, you can explain that you realized your results were not quantitative after you did the experiment, and you can describe what you would do next time to get quantitative results. It's important to communicate that you understand the process for doing a scientific investigation and what you've learned in doing the project.

Let me know if you have any questions about doing the write up.

Donna

[procrastinationking]

Thanks again, and yeah, camp was really fun I hope you know how much I really appreciate your help .

So far, I've finish my write up (the one that explains how my experiment failed, etc.) and since the project isn't due until next week Friday, I'm trying to think of what I can do now to get data. Right now I have an idea but I'm not sure if it's good. I would greatly appreciate ideas. Okay, so I've been taking pictures of my plates and since on the first and second day, when I used the Chlorox and antiseptic (Germ-X) didn't made the colonies disappear I didn't bother counting them.

But, since I just wanted to check, I looked at the pictures and counted. What I found was that more bacteria colonies grew even though I left the antiseptic covering the agar. This makes me think that these are resistant strains of bacteria so I'm thinking I'm going to remove the antiseptic, then reapply antiseptic and count how many more bacteria colonies grow. Hopefully, if my hypothesis is correct, more bacteria strains each time I treat my petri dishes (I count all the colonies and subtract the number of colonies that were there before the most recent experiment to see how many new ones there are) and I can use a line graph to represent my data.

Would that be a good data?

[donnahardy2]

Hi,

It's good that you have your write-up done. And yes, you do have time for one experiment that will hopefully give you some measurable data. Putting antiseptic on a plate of colonies, and then looking for the appearance of new colonies is a good idea, but won't really give you the data you need. Here's my suggestion for an experiment:

Materials: You will need sterile cotton swabs, sterile water, and 4 unused nutrient agar plates.

1. Designate a surface that hasn't been exposed to any chemicals for a day. This could be a kitchen counter where raw food has been prepared, for example.
2. Mark off an area of the counter that you can measure in square centimeters ( 10 x 10 cm, for example)
3. Using a sterile cotton swab dipped in boiled water (so it will be wet) wipe it over the surface you will be testing, and then wipe it evenly over the surface of an agar plate.
4. Put your two disinfectants on the surfaces you have just tested.
5. Repeat the procedure with the cotton swab, using exactly the same technique you used the first time.
6. Turn the plates upside down so moisture won't accumulate on the agar surface, and incubate for at least two to three days. Tape the two sides of the agar plate together so they can't accidentally be opened and exposed to new bacteria. Incubate the agar plates at 20 to 30 degrees Centigrade. (The warmest place in your house.) Use a thermometer to measure the temperature and record this information.
7. Count the colonies in each plate as soon as they are visible, which will be 2-3 days, depending on the temperature.

This experiment will give you a before and after count. You can graph the results using a bar graph and include in your results section.

Let me know if you are not able to get the additional materials, and I'll try to think of something else to do.

Donna Hardy

[donnahardy2]

Hi,

One more thing. If you haven't already, click on the "Science Fair Project Guide" on the top of this web page, and check out the information on microbiology on the Science Buddies website. There is good information on basic techniques and important safety information for working with live bacteria.

Donna

[procrastinationking]

Thank you very much for your suggestion.

Materials: 4 unused nutrient agar plates.
...4. Put your two disinfectants on the surfaces you have just tested.
5. Repeat the procedure with the cotton swab, using exactly the same technique you used the first time. ...

So this would mean that so far, I have used 2 plates, right? Then would that mean after 2-3 days I repeat step "4. Put your two disinfectants on the surfaces you have just tested," and "5. Repeat the procedure with the cotton swab, using exactly the same technique you used the first time?" And then again after 2-3 days? That way in the end the 4 plates are used.

[donnahardy2]

Hi,

I think you are using two disinfectants. Right? I was thinking about a control plate (before disinfectant) and after for each disinfectant on each section of surface that you test. If you are just using Chlorox, then you could get by with two plates as a minimum. However, if you have lots of plates available, then add two more control plates (one incubated without touching it to verify that your agar was sterile, and one inoculated with a known source of viable bacteria to verify that your agar would support bacterial growth). And if you have the plates and time available, then do your experiment in duplicate (two plates for each test). Science fair judges are always impressed when students in your age group run experiments in duplicate.

Please let me know about your results.


Donna Hardy

[procrastinationking]

Hi,

Sorry I wasn't clear before but I'm only using one disinfectant. Instead of doing it in duplicate, would it be okay to wait 2-3 days after applying the disinfectant to then apply it again and collect bacteria on another dish, and do the same thing 2-3 days later? That way I can test the resistance of the bacteria. (i.e. hopefully the first plate that isn't treated has the most, the 2nd plate that is the first treated has the least, the 3rd plate where the square is treated 2 times has a little more colonies, and the 4th one has more than the 3rd plate.

If that's not okay, then I'll do my project in duplicate like suggested. Currently I have 2 plates growing bacteria (untreated and treated).

Please let me know about your results.

I'd be happy to let you know my results but please don't get too upset if I don't reply right away. I usually get "sorta" grounded after a project since I stay up too late, so that means no internet. Haha . But I'll reply.

[donnahardy2]

Hi,

It sounds like you are thinking about testing for the development of resistance of bacteria to your antiseptic. This type of experiment would be a completely new project, and I would not recommend doing this unless you have the time. I thought you had completed your project, realized your experimental design was not measuring what you wanted to do, and were now considering one additional experiment to verify this. If you project is due in the near future, I would just use the results from the two plates that you have growing, the treated and untreated, and concentrate on our write-up. You have lots to discuss even though your initial experiment was not successful.

Is anything growing in your two plates? Can you count the individual colonies?

Please don't stay up too late. We need to finish this discussion so you can turn in your project.

Donna Hardy

[procrastinationking]

Yes, I have completed my write-up (except for this new experiment) and it was focusing mainly on antiseptic and bacterial resistance. It was during my second post that I switched from Chlorox resistance to Germ-X/antiseptic resistance. I'm sorry that I wasn't clear on that.

There is 52 colonies in plate 1 (untreated) and 12 in plate 2 (untreated).

Since these two plates are done I'm going to do this.

Use new, sterile cotton swab dipped in boiled water to wipe over the 10 x 10 cm square then streak over new agar plate. Then apply the antiseptic over it, same amount as the first time, wait 30 seconds [did this the first time (Germ-X says it kills many common germs in as little as 15 seconds)], use a new paper towel (dipped in boiling water and squeezed with sterile gloves) to pick up the antiseptic (cover the square get it off the square like popping a pimple or something *bring the antiseptic to the center then pick up*). Then use new, sterile cotton swab dipped in boiled water to wipe over the 10 x 10 cm square then streak over another new agar plate. I plan to repeat this process again after 2-3 days.

With the data recorded I plan to do this: (English is my second language so I'm not really sure how to word this) Find the percent of bacteria colonies of the untreated to the treated. For example, Trial 1 is 12/52 = 23.08%. If the bacteria colonies of the untreated dish is a closer percentile of the untreated after each trial, then that would mean bacterial resistance has grown.

Otherwise I can do the percentage killed (100 - 23.08 = 76.92%) and see if the percentage grows smaller each trial. But I'm not sure if the results can tell me that.

I'm still planning to include my first failed experiment except rework it to make it fit my project. I've been continuing to do it alongside the other experiment although now its using antiseptic. What I did is apply 15mL of Germ-X every three day to the dishes (leave in the antiseptic till the third day and get it out with sterile gloves, then apply new antiseptic right away). Every three days I count how much new colonies show up. Then I use that as data.
I don't really mind if I stay up late. It's common for me to sleep around 11:30-12:XX. Staying up too late means <3 hours of sleep.

My presentation is on Friday, and I don't need to have all my results (according to my teacher). I can turn that in on Tuesday. The actual science fair isn't until two weeks from now.

I apologize if I have made you feel you wasted your time.

[donnahardy2]

Hi Ellie,

Thanks for the additional explanation. I would never have guessed that English is your second language from your correspondence. Your English is excellent!

Your project is complete, and you really don't need to do another experiment. Your presentation is tomorrow, and the project is due on Tuesday, so you should concentrate on the oral and written presentations rather than do more experiments. Communicating results is as important as doing the experiments.

1. You are applying antiseptic directly to the agar surface, and then looking for the growth of bacteria on the agar surface. This experiment tests for the ability of colonies that are forming on the agar to survive, but does not give you a quantitative result. To measure the resistance of bacteria, you would need to start with individual microorganisms, expose them to the antiseptic, and then grow them on the agar to count them.

2. Two experiments are not enough to demonstrate the development of resistance, and your experimental design will not test for this. You would need to select colonies that survive the antiseptic, culture them, and then design experiments that would demonstrate increasing antiseptic resistance. This is really a whole science fair project that would be good to save until next year. If you do go ahead and set up another experiment, do it in duplicate so you can get an idea of the reproducibility of your technique. Setting up experiments twice, rather than one time, is better scientifically.

3. Scientists like to measure things, so you should try to measure the volume of antiseptic you use. If you do the experiment again, you need to try to add the same amount of bacteria to each plate. I'm not sure from your first experiment where the bacteria came from.



Here are some comments on your write-up.

1. Your approach to the analysis of your results is correct. If 52 colonies are present on the untreated plate, and 12 on the treated, then you observed a 77% reduction in colonies. You should not use decimal points because your results are not significant to .01%.

2. You changed from Clorox, which contains sodium hypochlorite, to Germ-X, which contains ethyl alcohol (ethanol) and other ingredients:

Active Ingredients: Ethyl Alcohol (62%) (Antiseptic)

Inactive Ingredients: Carbomer, Fragrance, Glycerin, Isopropyl Alcohol, Isopropyl Myristate, Propylene Glycol, Tocopheryl Acetate, Water

Did you include information about how ethanol kills bacteria? Here is a website that contains information about various bactericides:

http://www.umsl.edu/~microbes/pdf/disinfectants.pdf

This website explains how ethanol kills bacteria:

http://www.parish-supply.com/what_reall ... _germs.htm

Do you have any other questions about your write-up? Have you included every section on the outline your teacher gave you?

Don't worry about wasting my time at all. Science buddies is a resource that you should use whenever you need help with a science fair project, and I hope my comments have helped you. Next year, write us a little earlier, and we'll give you suggestions that will help you design your experiments.

Good luck on you presentation tomorrow!

Donna Hardy