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

[procrastinationking]

Hello again,

Kind of good news is I ask my teacher if I could present on Tuesday because I told her I didn't have all the results, yet. Not sure if it's good since my experiments don't test what I'm looking for(?).

...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.
...You would need to select colonies that survive the antiseptic, culture them...
3. ...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.

Does individual microorganisms mean individual bacteria cells? If they are how would I expose them to the antiseptic? If they are just the bacteria colonies growing on the agar, how would I expose them to the antiseptic? The same way as the first experiment ("pour" it in and let it stay?)?
Also, since this is my first time growing bacteria I'm not sure how to move bacteria colonies to another dish to culture them (I wouldn't need to know which ones are alive do I?). Do I just wipe over the agar surface with the bacteria and then streak a new plate? How would I be able to add the same amount of bacteria to each plate? The bacteria from the first experiment came from a trash can lid, though it wasn't a measured area.

This experiment tests for the ability of colonies that are forming on the agar to survive, but does not give you a quantitative result.

How would I explain why the results aren't quantitative. Would they be qualitative? Also, I'm not sure if I should mention this, but my results for one of the plates are:
Untreated: 142; Treated 1x: 146; Treated 2x: 156; Treated 3x: 172. This technically means that 4 colonies grew with the Germ-X covering the agar for three days the first time, 10 the 2nd, and 16 the third. The colonies are counted 3 days after treated with Germ-X.

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%.

What type of results will this experiment give? And if I did do:

Since these two plates *regarding the experiment you suggested me 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 *same square* 10 x 10 cm square then streak over another new agar plate.

would it still have nothing to do with bacteria resistance? If yes, then what exactly is my experiment testing? Bacterial reduction/how well Germ-X works? Would my discussion and conclusions be something vaguely like: The experiment has failed in providing results that would be able to determine if bacteria has developed resistance?

Also, is it necessary to include the inactive ingredients of Germ-X in the materials or discussion? And, yes, when I changed from Chlorox to Germ-X, I included how ethyl alcohol kills bacteria. Thank you for your concern and for your effort to me sites just in case I didn't.

The presentation tommorow and Tuesday is the class presentations. The actual judging isn't until the Thursday two weeks from now (although that doesn't mean that the class presentation isn't important). I think I have more to say but I lost whatever it was.

EDIT: Are the result of the experiment you suggested quantitative?

P.S.

Hi Ellie,

I think you've gotten me mixed up with someone else. My name is Andi (My mom thought it would be unique T_T to have Andy spelled differently). Okay, now that you know what to call me, how should I address you?

Sorry if my grammar isn't as well as before. I usually take about an hour to make my post and half an hour to rework the grammar (as best as I can). However, I need to work on my board now (cutting out, pasting, etc.)

[donnahardy2]

Hi Andi,

I'm sorry I was using the wrong name. Andi is a unique name, and I'm glad you told me I was using the wrong name. You can call me Donna.

OK. You have time to do one quick experiment if you set it up today. There's just enough time for colonies to grow by Sunday or Monday. Individual bacteria are usually about 1-2 microns in size, so are only visible under the high power of a microscope. One way to count living bacteria is to transfer them from wherever they are sitting to an agar plate and let them grow into colonies. It is assumed that one colony on a plate represents one bacterium from the original sample. A visible colony contains a minimum of one million bacteria.

The reason that your original experiment didn't give you measurable results was that you started with colonies containing millions of bacteria, and then applied the antiseptic. There was no way to measure the effect of the antiseptic because you didn't know how many individual bacteria were present before the experiment.

For your new experiment, you won't be starting with the agar plates you already have. You will be using bacteria that are naturally present in your environment and hopefully the number will be low enough to count. Using the trash can lid again would be an excellent choice, but use a measured area such as 5 or 10 square centimeters to test. You will choose a surface, and wipe a specific area (measured in square centimeters) with a sterile moist cotton swab, and then wipe the swab on the agar surface. Then you will put the antiseptic on the same surface, and repeat the process.

For your experiment, look back at the procedure I wrote on the 18th. This experiment will measure the number of bacteria on a surface before and after antiseptic treatment, and will give you the measurable result you need, because you will be starting with individual bacteria that are on the trash can lid or whatever surface you select. You will be using the antiseptic on the surface you are testing, and not putting it on the agar plate. If you have 4 agar plates, then set up your experiment twice, using another surface like the bathroom floor or a door knob. If you have 2 more plates, then use one as a negative control (to show that your agar plates were sterile), and one as a positive control (transfer colonies from one of your original plates with a cotton swab to show that the agar would support the growth of bacteria). You will report your results as number of bacteria/square centimeter before and after. And yes, your result will be quantitative. The results can be shown in a bar graph.

Answers to other questions: Individual bacteria are the same as individual microorganisms. Microorganism is a more general term and could include other small microbes such as fungi.

In your discussion, you could say that your original experiment did not show that bacteria had developed resistance to the antiseptic because you realized it was not a quantitative experiment. If you get results on your second experiment you will be able to say that the bacteria you tested were sensitive/resistant to the antiseptic you used. If the bacteria are resistant to the Germ-X, you won’t know how they developed resistance because your experiment didn’t test that.

Your experiment will measure the effect of Germ-X (ethanol plus the other ingredients), so you should mention that Germ-X contains other ingredients. You really don’t know what the result of using pure ethanol would be from your experiment. You could include the inactive ingredients in Germ-X in your discussion, and perhaps suggest that it would be interesting to measure the effect of ethanol alone or Germ-X (ethanol plus the other ingredients) to see if the other ingredients are really inactive. (Possible topic for next year’s science project, not this week-end).

Your project, either with or without the new experiment is a complete project. You set up an experiment, and observed some results. The results of the original experiment helped you design a better experiment, and this is what using the scientific method is supposed to do. If your teacher is requiring quantitative results for the project, then this week-end’s experiment will give you quantitative results. Please note that if nothing grows on your plates, your results will be zero, which is a number, and still counts as being quantitative. If too many colonies grow on the plates, then microbiologists count this as TNTC (too numerous to count), which is usually >300 (or whatever you can count), and this number will be considered quantitative also.

Please let me know if you have any other questions. And do let me know what your teacher says about your presentation next Tuesday.

Donna

[procrastinationking]

Hi Donna,

For your experiment, look back at the procedure I wrote on the 18th. This experiment will measure the number of bacteria on a surface before and after antiseptic treatment, and will give you the measurable result you need, because you will be starting with individual bacteria that are on the trash can lid or whatever surface you select. You will be using the antiseptic on the surface you are testing, and not putting it on the agar plate. If you have 4 agar plates, then set up your experiment twice, using another surface like the bathroom floor or a door knob. If you have 2 more plates, then use one as a negative control (to show that your agar plates were sterile), and one as a positive control (transfer colonies from one of your original plates with a cotton swab to show that the agar would support the growth of bacteria). You will report your results as number of bacteria/square centimeter before and after. And yes, your result will be quantitative. The results can be shown in a bar graph.

I did two experiments. The first one is the one where I applied the antiseptic to the agar plate. The second one is the experiment you suggested on the 18th. I did it the exact way the procedures said (kitchen counter). I applied the antiseptic to the kitchen counter area after collecting the individual bacteria on the surface. Then repeated the process (but I had to remove the antiseptic first). That got me my results:

...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%.

, which means 52 individuals in the first dish and 12 individuals in the second dish. Would this be my quantitative results, or should I do a new experiment again? Also, I currently have two 3-day-old dishes in which one has the bacteria from the same surface as the first two (kitchen counter top square).

This means I have 4 dishes if I'm to decide they belong in the same experiment. So dish 3 is the dish with the bacteria transferred from the countertop after 3 days of applying the antiseptic. The dish 4 is the dish with the bacteria transferred from the counter top again after applying antiseptic to the surface again, after collecting the bacteria for dish 3( same amount of antiseptic as the first time). Right now they have no colonies. However when they do, suppose dish 3 has 30 colonies and dish 4 has 15 colonies (just hypothesizing here). This would mean there was only a 50% reduction.

If and dish 1 and dish 2 is trial 1 with a 77% reduction, and dish 3 and 4 is considered trial 2 with a 50% reduction, then can I conclude that bacterial resistance was developed? If so, then would it be [50/77=65% --> 100-65=35 (is this how you find resistance?)] the bacteria developed 35% resistance? Of course, I still haven't seen any bacteria grow yet so there may be no resistance whatsoever or this experiment might not test resistance.

Thank you very much for your help.

[donnahardy2]

Hi Andi,

Thanks for confirming exactly what you did on your experiment. Here's what you can say about your results:

Dish 1 and 2: You measured the number of bacteria on the kitchen counter before and after applying the antiseptic. You observed 52 colonies before antiseptic and 12 colonies after, so you saw a 77% reduction in the number of colonies.

Dish 3 and 4: You measured the number of bacteria on the kitchen counter before and after applying the antiseptic, so you repeated your first experiment. The bacteria that were present on the kitchen counter for the second experiment were not the identical bacteria that were present the first time. Hopefully, the colonies on these plates will grow up and be visible by tomorrow. If they do, you can make the same calculation and quantitate the results. If no colonies grow, then you will have observed 0 colonies before and 0 colonies after antiseptic. If you do see colonies, then you will have some positive numbers and can make a calculation.

Development of resistance: Your experiment did not measure the development of resistance to the antiseptic. Your experiment measured the resistance of the bacteria that were present on the kitchen counter at the time you did the experiments. To measure the development of resistance, you would need to take the bacteria from dish 2 (the 12 colonies that survived the Germ-X) and expose then repeatedly over several generations and design a new experiment that would measure an ability of the bacteria to grow when exposed to increasing concentrations of antiseptic. Your experiment is a good experiment, and the science fair judges will be completely impressed that you actually repeated your experiment, so do not worry that your experiment did not do what you originally intended.

Conclusion: Based on your first experiment, your conclusion is that 23% of the bacteria on the kitchen counter were resistant to the Germ-X. (You can revise this number if colonies grow on dishes 3 and 4.)

Discussion: You have several topics for your discussion section:

1. Resistance to Germ-X: The manufacturer of Germ-X claims that it will kill 99.99% of microorganisms that cause disease. If this were true, then you would have expected 0 colonies on dish 2. You used the Germ-X according to directions, so why did the bacteria grow? Perhaps this means that the 12 colonies were not bacteria that caused disease (most bacteria do not cause disease), or maybe you didn't test the Germ-X the same way that the manufacturers did when testing their product claim. Or, maybe you purchased a bottle of Germ-X that did not contain the correct amount of ethanol, or maybe the product claim is not true. You would need to do more experiments to find out why the Germ-X did not work, but this is a very interesting observation.

2. Reproducibility: The results from dishes 3 and 4 will give you an idea about the reproducibility of your experiment. If you see similar results with the two new dishes, then you can say that your experimental design was good, and you will have confidence that your conclusion is correct. If the results are not similar, then you will be less confident.

3. Development of resistance: Since your experiment just measured resistance at one point in time, you didn't achieve your original objective of measuring the development of resistance. You should present a couple of your own ideas here about what additional experiments could be done to show the development of resistance over time.

4. Second experiment: If no bacteria grow on dishes 3 and 4, then you need to suggest reasons for this result. Perhaps your agar plates had dried out, perhaps the temperature was not optimum for growth, or perhaps there was not enough time for colony formation before your deadline.

This is a really good project, and you are almost done! Let me know about the results of dishes 3 and 4, and if you have any last minute questions.

Donna

[procrastinationking]

Hello again,

To measure the development of resistance, you would need to take the bacteria from dish 2 (the 12 colonies that survived the Germ-X) and expose then repeatedly over several generations and design a new experiment that would measure an ability of the bacteria to grow when exposed to increasing concentrations of antiseptic.

I know I don't have time left but I'd like to know, how would I take the bacteria from dish 2 and expose them and then to a different dish without an innoculating loop? To transfer bacteria from one medium to another, is it possible to use a sterile cotton swab and wipe over the surfaces of where the colonies are on dish 2 and then streak it onto a new dish? Also, is the way to expose them to the antiseptic the same way I did in my first experiment (apply antiseptic to the agar plate)? These questions are just so I can design a better, new experment for next year.

Conclusion: Based on your first experiment, your conclusion is that 23% of the bacteria on the kitchen counter were resistant to the Germ-X. (You can revise this number if colonies grow on dishes 3 and 4.)

Unfortunately I have no colonies for dishes 3 and 4. How would I put this data into a graph?

Thanks again.

EDIT: My title is "Antispetics: What Happens to the 0.01%?" because on antispetic labels it always say kill 99.99% of bacteria. However, since My experiment is not testing resistance, what should my title be? (My background information is about resistance, though.)

[donnahardy2]

Hi Andi,

No, you don't have time to do another experiment, but if you are interested, you could start working on this project for next year. You would need an experiment that would allow you to grow up bacteria so they could be counted, then add different concentrations of antiseptic, and then count the survivors. By selecting the survivors from each experiment, you could see if any would survive with gradually increasing concentrations of antiseptic. But please don't worry about the details of the experiment today, just concentrate on finishing up this project.

Graphing two data point that are "zero" will not make a good graph. Just make a bar graph titled "number of bacteria" and make 1 bar representing 52 and label it "before antiseptic" and another bar representing 12 and label it "after antiseptic." In your text, write a sentence explaining that you repeated the experiment and there were no bacteria that grew on either plate. You will probably want to say that it is unlikely that there were no bacteria on the kitchen counter for your second experiment, but for some reason they didn't grow. Perhaps the incubation time was too short, or the agar was old and had dried out. You can include an explanation of the "zero" results in your discussion section.

Your title is great; it will catch the interest of anyone coming to the science fair, so don't change it. It is not unusual at all for projects to change directions once the experiment is started, so do not be concerned that your project did not accomplish its original goal. Your experiment did test the resistance of bacteria to the antiseptic that were on your kitchen counter, and you found that 23% survived, instead of the expected .01%. Your discussion should focus on explaining your results, (refer to my suggestions 1-4 from yesterday).

Reproducibility: Your experiment was not reproducible because the results of your first and second experiments were not the same. Your discussion should acknowledge that it would be a good idea to repeat the experiment to verify the results of the first experiment.

In your presentation tomorrow, do not use any phrase that would suggest that your project failed or did not accomplish your original goal. Your original goal was very ambitious, and probably not practical with the time and resources you had available. But, you designed an experiment, obtained results, and you can explain the project and discuss the results. You have a very complete science fair project to turn in, and I know you have learned a lot about your subject and doing scientific experiments.

Good luck!

Donna

[procrastinationking]

Hi Donna,

Thank you so much for your help.

In your presentation tomorrow, do not use any phrase that would suggest that your project failed or did not accomplish your original goal.

Haha I was planning on saying my experiment failed, but I guess I won't! But then, what about when the actual judges judge me (not the teacher)? My teacher grilled all the presenters on Friday and I think she'll get pretty mad if she finds out my experiment failed.

If I don't crash from making my board (which will cause me to not sleep), I'll be sure to post what happened. Hehe, my name is procrastinationking (although this is the project I've procrastinated the least on )


Thank you for all your help, although I think I'll still need it soon (Teacher's making us duplicate our experiments in class or improve it, if we have time).

[procrastinationking]

I'm such an idiot! I was going to post this yesterday in the edit, but I was afraid I would've made you think that I was an idiot.

If you have 2 more plates, then use one as a negative control (to show that your agar plates were sterile), and one as a positive control (transfer colonies from one of your original plates with a cotton swab to show that the agar would support the growth of bacteria).

Last night, when I thought about it, I found out why the bacteria didn't grow in my dish 3, 4 and these new dishes 5, 6. It's because I was an idiot and microwaved the agar solution instead of boiling it. Originally, I was stupid and boiled 10 ml of agar solution at a time. Of course, this would mess up a whole lot of petri dishes because most of the time the agar would harden before all of it was poured into the petri dish. So, I had this "bright idea" to try and microwave the solution (I read it somewhere online before) and so I microwaved it in a small 10ml beaker. This was much easier to pour into the petri dishes so I did so for dish 3, 4 , 5, 6. Stupid, I know. I changed a control thinking it wouldn't matter . So last night, with my two remaining petri dishes, instead of creating a positive and negative control, I created dish 7 and 8. But this time I boiled the solution to see if my "hypothesis was correct." By now, I learned the right way to prepare agar solution (make a big batch and distribute it evenly). I did the kitchen counter square (even though it hasn't been three days after doing dish 5 and 6), like before. Now, after just one day, there is 3 colonies in dish 7 and 2 in dish 8, while there is none in 6-days-old dishes 4,5 and 3-days-old dishes 5,6.

I didn't want to tell you last night about the microwaving because I was worried I would dissappoint you, but I just have to tell about it. I'm so sorry. If only I posted it yesterday, you would've guided me on what to do. Since I'll probably be staying up, I'll check this thread at 5 am (which is the time you usually reply due to the time zone differences). If theres nothing, then I'm not sure if I should include this blunder in my discussions.

I had a feeling the easier way would mess thing up


EDIT: I've got it. I know this may sound stupid but this is what'll do: In the procedures I put "repeat steps xx-xx every 3 days for 6 days. wait 1 day (because this is what I did for dish 7,8)" and steps xx-xx is the part where I apply the antiseptic to the square. Then I'll put in steps where you have to make two new dishes and collect the bacteria from the square and put it onto dish 7 (which I'll rename dish 3) and then apply the antiseptic and put it onto dish 8 (which i'll rename dish 4). Incubate until colonies appear. Right now Then I'll put compare the resistances between the two trials. 2/3 is a 66% resistance compared to the 23%

[donnahardy2]

Hi Andi,

One of the things I haven't told you is that last minute experiments almost never work. With science fair projects, it's usually better to stop one week before the deadline, no matter what, and concentrate on the write up.

Microwaving agar does not prevent bacteria from growing. If the agar was burned, then it might inhibit growth, but I don't think you know why the bacteria didn't grow on plates 3 and 4. The pH might be wrong or it might be too dry. It would take another science fair project to find out exactly why. And you are right, once you have a technique developed to do an experiment, it's better not to change it, because that can definitely affect the outcome.

It will be very confusing to viewers looking at your board if you try to explain your last 2 experiments (plates 5- 6 and 7-8). Just use the results from plates 1 and 2, and say that you could not reproduce the results by repeating the experiment with plates 3 and 4. You have a very low number of colonies growing on plates 7 and 8, and these might be contaminants. I think you should just present the results you have. You have a complete project the way it is. If you have already put the additional information in, since it is due in a couple of hours, then just leave it in. You will be able to explain to your teacher what happened. It's important to present information clearly and concisely on the board so readers can understand what you did.

I think you did a good job on this project, but I think you are too worried about what may have gone wrong. Nothing ever works out perfectly when doing research and your experience is very typical. Please try to relax and in your oral presentation today, remember that you have a lot of knowledge and experience to share with your class. You are the expert on your project and you need to communicate that to others.

Let me know if there's anything else I can do to help!

Donna

[procrastinationking]

Ehhh I finished my board at 4:11am (your post is at 4:19) and unfortunately I did the opposite of what you just told me. However, I did say my data for trial 2 was unreliable.

I'm too tired to change it since I still have other homework
I leave for school at 6:30 am.

Thanks for everything.

[donnahardy2]

Hi Andi,

Don't worry about it. Your board will be fine. You've finished your project!

Donna

[procrastinationking]

Today in class we took a quiz instead of presenting our project (our class is behind the classes that don't do science fair, that's why). So after school, we just showed her our board. I only got to talk to her for about 6 minutes (she had to leave early) during which I was going over whther or not I should tell her how my experiments failed to find the development of resistance. I didn't but I did tell her that I needed to change my experiment. She also told me to have at least 3 trials, preferably 5, which means I have to redo the data which will cause me to change my discussions and conclusion. I'm going to duplicate the experiment tomorrow in class but I'm not sure if I should use the same experimental procedure.

What do you think? Regardless, I'm going to make 2 agar plates tomorrow in class and collect bacteria samples. I would like to do an experimental procedure that tests the development of reistance but I have no idea how to do that.

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.

You would need an experiment that would allow you to grow up bacteria so they could be counted, then add different concentrations of antiseptic, and then count the survivors. By selecting the survivors from each experiment, you could see if any would survive with gradually increasing concentrations of antiseptic.

I still have no idea how to select survivors and culture them (cotton swab?)I'll probably end up just duplicating the experiment you suggested on the 18th since the judging is next week thursday (my teacher isn't sure if I'll have enough time.)

My board did not include my original experiment since there wasn't enough space.
Do you know what's a good way to take pictures of bacteria growing in agar dishes? I usually hold the dish up towards a light but my teacher told me there's a different way (she's not a biology teacher so she doesn't know).

I'm not sure if I should be happy about not presenting today (I did poor-quality work on my other subjects' homework due today), but at least I have more time to do another experiment(?).

Thank you for continuing to help.

[donnahardy2]

Hi Andi,

You have set up your experiment 4 times, so I don't understand why you need to set it up again. Colonies grew in two of the experiments, so you have results to report. Sometimes, as you have discovered, experiments don't work out as expected. But you need to do the experiment to find out what will happen.

At this point, since your science fair is next week, I would recommend not spending too much time doing the experiments. You absolutely don't have time to change your experimental design. So just set up the experiment outlined on the 18th; prepare the agar by boiling like you did originally. Count the bacteria by swabbing the surface before and after you apply the Germ-X according to directions. You can set up more than one set of before and after plates, choosing different surfaces if you would like. Your kitchen counter doesn't have many bacteria, so it would be nice to have a really high number on the before plates. If you set up the plates today, you should have colonies growing by Saturday.

I don't know the best way to photograph agar plates. I would imagine that putting the plates on a dark background and using indirect light so the shiny agar and colonies don't reflect the light would work best. But, I'll ask at work today and see if anyone has had experience in the subject. If your photographs are not clear, you can draw a picture showing the results, and this might be clearer to the people looking at your project board.

Your project is really about finding out if the bacteria you are culturing are resistant to Germ-X, and this is a perfectly good project. You need to make sure your board will communicate the details of your project clearly when you enter it in the science fair next week. If you have your project written in a word document, you could copy and paste into this board and I would be happy to make comments to help you.

I hope you can present your project today, so you can get that part out of the way. And, I'm sorry you had to neglect your other subjects, but that is unavoidable sometimes. But you sound like a hard worker, so I'm sure you will catch up soon.

Donna

[procrastinationking]

Hi Donna,

Gahh, my session timed-out so I have to retype everything (although it may be less detailed).

Thank you for your help.

Today I went in to school and finished up my experiment (although I have to wait for the bacteria to grow. What I did was have new dishes 1a-1c and 2a-2c collect bacteria from a dusty area that doesn't get clean often, the teacher's desk for 3a-c and 4a-c, the bathroom doorknob for 5a&b and 6a&b, and the classroom doorknob for 5cand 6c. For the dusty area and teacher's desk, I made 3 adjacent 10x10cm squares (although I drew each square only after I finished the process of the first square) and did a before and after.

Should I include the doorknobs in my results or would it be strange (since the bathroom doorknob 5a&b and 6a&b are the same doorknob while 5c and 6c are from a totally different doorknob, the clasroom one.)

Also, the positive control...I'm not really sure how to do it. I have the plate but I didn't streak anything onto it.

one as a positive control (transfer colonies from one of your original plates with a cotton swab to show that the agar would support the growth of bacteria).

What is the process to transfer colonies? streak the original plate and then streak the positive control? And what does "original" in original plate mean?

Another question is: should I include the results from my kitchen counter?

Thank You,
Andi

P.S.
Since I have to wait for my bacteria to grow to revise my data and discussions and conclusions, I'll just post the background research.

What are Antiseptics?
An antiseptic agent hinders the growth (bacteriostatic) of or kills (germicide) microorganisms on the external surface of living objects. They are different from antibiotics and disinfectants because antibiotics kill microorganisms inside the body, and disinfectants destroy microorganisms on non-living objects. Antiseptics are also known as skin disinfectants.
History
Sir Joseph Lister (1827-1912), an English surgeon, was the first to introduce the idea of antisepsis. After learning about the germ theory of disease as well as having a high mortality rate due to infections after surgery at the Glasgow Infirmary, he reasoned: if infections were caused by microbes, then to prevent an infection the best way would be to kill the microbes before it reaches the open wound. He employed the use of carbolic acid, or antiseptic phenol, as an antiseptic agent in his work and the acid, along with other antiseptic procedures he instituted, dramatically decreased postoperative deaths. Soon, every hospital applied his techniques.
Use

Joseph Lister developed antiseptics for use in the hospital. However, during the mid-1990s there were over 700 household products containing antibacterial agents. Although most are used for cuts, hand sanitizers and hand lotions are being overused.
How Antiseptics Work
Most antiseptics work by destroying or damaging the cell wall of bacteria. There are many different groups of antiseptics but the performed experiment focuses on the alcohols group. Mainly ethyl alcohol and isopropyl alcohol are used as antiseptics. It is believed that they cause rapid denaturation of proteins causing interference with metabolism and cell lysis. It is also believed it causes membrane damage of the cell. However, antiseptics, like disinfectants, don’t kill all the bacteria.


Resistance
Scientists are worrying that the overuse of antiseptics, along with disinfectants, are causing bacteria to develop resistance to the antibacterial agent aimed at it, and possibly other antimicrobial agents as well (cross-resistance).
A former high school student, Merri Moken, recently did a study on if disinfectants creating mutant bacteria strains resistant to antibiotics. Antibiotics and disinfectants kill germs by inhibiting protein synthesis or by breaking down bacterial cell walls. Because the disinfectant killed bacteria the same way as antibiotics, when she treated her bacteria cultures, the surviving bacteria were the ones resistant to the disinfectant. She later tested if they were resistant to antibiotics, which they were. This is due to gene marA which is over-active in antibiotic-resistant bacteria.
Some disinfectants/antiseptics cause microorganisms to become resistant to them by leaving behind a residue or film on the surfaces it were applied to. The bacterium that survived during the application will reproduce and multiply in the residue. These new strains will be more resistant to the disinfectant/antiseptic, making them almost useless.

[donnahardy2]

Hi Andi,

I'm sorry you had the retype everything after you timed out. I know that is frustrating.

Your background research section is exceptionally good, one of the best I have seen lately, and it is a perfect introduction to your experiments. I don't really have any suggestions because it is very complete. If I were judging your project, I would be convinced that you did a thorough job in doing the background research. I assume that you have references cited in a bibliography section to document the sources of your information.

To investigate the resistance of bacteria to the Germ-X, you are testing the resistance of bacteria in your environment. You won't know if the bacteria, which grow after exposure to the antiseptic, contain the marA gene, because you did not test for it. However, you will know if there are antiseptic-resistant bacteria present because they will grow after exposure to Germ-X.

If you see growth on any of the plates, you won't need to worry about a positive control. The positive control verifies that the agar has the ability to support the growth of colonies. If you see zero colonies on all of the plates, then you will know that you should have included the positive control. The plate that you kept without streaking anything is a negative control. It is a test to verify that the agar was not contaminated with bacteria before the experiment. If you see colonies on the negative control, then you won't know if your results are valid or not.

When your colonies grow, count all of the plates. Include the original kitchen counter results, and all of the before and after sets, even the 5 a&b and 6 a&b, even though they are for the same door knob. For your results section, use a bar graph and graph every set of results, using one color for the "before", and another color for the "after" results. I am a little confused about your numbering system and I don't understand where 5 c and 6c came from. If 5c was a test of the class door knob before antiseptic, and 6 c was a test of the same door knob after antiseptic, then this would be a good set of data to include. Please verify that 1a and 1c (and the other a and c sets) were a count of the bacteria of a surface immediately before and after antiseptic treatment. If they are not, then please tell me about the timing of these tests, so I can understand how you set up the experiment.

Do you have any other sections to post?

Donna