Science Buddies: "Ask an Expert"

Hi Everyone. I'm hoping for some advice despite my lame subject line

The original plan for the science fair this year was the experiment from your microbiology section - Do Different Dilutions of Disinfectants Affect the Development of Bacterial Resistance?.. using E coli and Lysol. Well, that was the plan until the instructor said that the topic was way overdone. So, my niece (more of a history kid ) and I were thinking along similar lines.. basically substituting the bacteria obtained from a "regular" grocery store chicken (having antibiotic exposure) and an organic one(no antibiotic exposure) for the E coli and the antibiotic ampicillin for the disinfectant.. then comparing the resulting zones of inhibition. I don't know though..does this have any value or worth at all? If by some chance it does, I'm not sure how to handle the ampicillin issue. In one experiment, the procedure calls for 3 different amounts of antibiotic to be tested ( 1mg, 3mg, and 5mg ampicillin powder to be added to 10ml water.) However, I'm not sure she will have access to the type of scale she needs to measure these amounts. Then, on a science supply site, I found Ampicillin Solution 100 mL (product description being 1 mL of antibiotic solution per 100 mL of media). If we were able to order this product, what differing amounts/strengths of the antibiotic solution would you suggest we use on the discs placed on the bacteria?

I would appreciate any thoughts/advice/suggestions for alternatives or revisions. Thanks so much for your time. You guys provide a really valuable service here!

Hi!

Your project is definitely important and your additions are some interesting ones that have not been tested before. Testing the difference in the resistances of antibiotically treated chicken bacteria vs organic bacteria is definitely a solid start. You might consider changing the idea of ampicillin as the disinfectant as it only affects certain types of bacteria called gram positive bacteria and very few gram negative bacteria. Using different concentrations of lysol isn't a bad starting off point either.

As for the milligram amounts, you could make 1 liter of solutions, which means you would be 0.1 gram, 0.3 g, and 0.5 grams of ampicillin in 1 L of water, which definitely doable in any science lab at school.

Hope that helps!

-Sam

Hi Sam,
Sorry to be so late in my response, but we really appreciated your input! She turned in her question and hypothesis yesterday, so she is on her way. We may be back for more advice though. Thanks again.

Very cool ideas about your science fair project. It is a very interesting and relevant topic, but I wanted to point out a few things to be aware of. First of all, how are you going to get the bacteria off of the chickens? You could probably use a sterile swab and then transfer the bacteria to an agar plate. Once there, you will have a few other things to think about. First of all, what if you find huge differences in the total number of bacteria cells from each kind of chicken? Second, it is well known that chickens often contain Salmonella bacteria, which can cause you get sick if you ingest it. Therefore you will want to be cautious about exposure to the cells that you culture off of the chickens. My suggestion would be to transfer the bacteria from the chickens to 4 different plates. One that has no antibiotic and one that has a low amount of ampicillin. That would allow you look at the % reduction instead of dealing with the overall numbers of bacterial cells. Also be sure to not open the plates after you have cultured bacteria off the chickens. I can't wait to hear about your results!

Matt Badtke

Hi. I'm finally back again. Our plan was to cut up chicken parts from the non-organic and organic chickens and put them into flasks containing 100 ml of sterile water. After letting them sit for 10 minutes, sterile swabs would be used to collect samples to spread across the surface of the agar. Then she would place Lysol infused disks inside the plates and hopefully zones of inhibition would develop. Is this not going to work well? Thank you. We appreciate any guidance you can give.

Hi,

This is a great project idea.

I thought you were going to use ampicillin. If your hypothesis is that the bacteria from chickens that are raised on antibiotics are going to have a greater resistance to antibiotics, then switching to a product containing a quaternary amine, is probably not going to work as well as you would like. Let me know if you need more explanation on this.

Antibiotic resistance is usually measured using a pure culture of bacteria that are freshly grown and then diluted to a consistent concentration before being applied to the surface of an agar plate. This ensures that a consistent quantity of actively growing bacteria will be used for testing. And your results will also be more reproducible. Since you will be using a mixed culture of bacteria directly from the chicken, you could collect the bacteria in a sterile test tube and dilute them until all of the samples and control have about the same turbidity. A barely turbid sample will contain about 10,000,000 bacteria per ml, which is perfect for making a lawn of bacteria.

The bacteria on the chickens will be primarily on the surface of the meat. If you are going to start with whole chickens, you could also consider rinsing the body cavity with 50-100 ml of sterile water, and collecting the water to transfer to the surface of an agar plate. This would be easier than cutting up the chicken. Whatever method you use, you should make sure you use the same technique with every chicken.

What are you going to be using for a control?

Donna Hardy

Hey. Thanks for your response.

We were thinking about antibiotics at first, but then we thought there might be some difficulty obtaining them so we went back to Lysol. The idea for the procedure (which I'm nervous about now..hope we haven't just totally missed the mark) involves running ten trials each of 100%, 50%, 25%, and 12.5% of Lysol (through disk diffusion) against the bacteria obtained from one organic chicken and the same ten trials against the bacteria obtained from one non-organic chicken. My idea of control plates are just bacteria from the organic chicken on one and the bacteria from the non-organic chicken on the other to show that the agar can support the uniform growth of bacteria. The hypothesis is that the bacteria from the non-organic chicken would be more resistant to the Lysol.

Just to make sure I understand, are you saying that rinsing the body cavity with the sterile water and then capturing it for use on the agar should be sufficient for growing a lawn of bacteria on the agar surface? Also, since this experiment is not very involved, do you think it would work to do an extension in which ampicillin disks (which we found online after we went back to Lysol) could be tested against both types of bacteria? In the same vein, I tried thinking of how to involve the gram negative/gram positive angle in an extension project by ordering some antibiotic disks that work against gram positive and some that work against gram negative, but since the bacteria on the chicken are of unknown type to us, would this be a useless exercise?

I really appreciate your help; I know these questions must seem really simple and silly, but I enjoy working with Lauryn on these projects and learning new things. Thanks.

Hi,

I think it would be a better experiment if you could get two different types of antibiotics. Antibiotics usually have a defined mechanism of action and your idea of comparing results of antibiotics that are effective against Gram-negative and Gram-positive microorganisms is a really good one. Lysol contains cresol, a disinfectant that kills bacteria when present above a certain concentration. Bacteria don’t develop resistance to disinfectants, so I don’t think your results would be as interesting with Lysol.

So I encourage you to try to get the antibiotic discs. Try to purchase enough disks of one lot of antibiotic to ensure having disks with a consistent concentration so you can compare results from one experiment to the next.

What you are planning to use for controls?

Donna Hardy

Hi. Thanks for the quick response.

So.. then maybe .. Bacteria from organic chicken swabbed on 20 plates.. A Vancomycin disk(effective against Gram-positive bacteria) in 10 of the plates and a Streptomycin disk (effective against Gram-negative bacteria) in 10 of the plates. And repeat the same for the non-organic chicken? And would the Johnson and Case table of values used to evaluate bacterial response be the correct one to use ( diameter of 10 mm or less means resistance, etc.)

Controls? I'm a little confused. In a prior experiment when she tested green cleaners, she was instructed to swab bacteria onto the surface of agar just to ensure that the plate could support uniform growth of bacteria and that was called the control plate. So, two control plates, one inoculated with bacteria from organic chicken, and the other with bacteria from non-organic chicken? Or maybe with a disk dipped in sterile water inside each one? We have come up with controlled variables like same techniques used, same place and length of incubation, but I'm thinking you mean something else?

I appreciate your patience.

Hi,

I recommend doing a small pilot experiment and testing just 2-3 plates to make sure your experiment will work first. Since this is an original protocol, you may find that you need to make some adjustments in the procedure. For the main experiment, using a larger number of plates would be excellent.

In addition to the negative and positive growth control, which confirms your agar is sterile and is suitable for growing chicken bacteria, it would be good if you could include a known Gram-negative and Gram-positive test organism with known antibiotic resistance to verify the antibiotic disks are working and that the test procedure is valid. Your experiment will still be good if you can’t do this, however. You can use the Jonson and Case table values as a reference, but since you aren’t using the standard method (pure culture of test organism in log phase of growth diluted to a standard concentration), the numbers may not apply. You can measure the zones of inhibition to get quantitative data, but you will want to compare results of organic and non-organic chickens when you evaluate your results.

I hope this helps.

Donna Hardy

Hi again,

So for one possibility for the controls.. obtain S. epidemidis (Gram-positive bacteria) and E. coli (Gram-negative) bacteria cultures from a science supply site and use these two to inoculate agar plates. Then put an Erythromycin disk (effective against Gram-positive) and a Streptomycin disk(effective against Gram-negative) in both plates. The expectation would then be that the Erythromycin disk would have created a zone of inhibition in the S. epidemidis plate , but there would be no zone of inhibition around the Streptomycin disk in that same plate, and the opposite situation would exist in the E. coli plate? And using disks from the same magazine would mean that if the Erythromycin is effective against a known Gram-positive bacteria (S.epidemidis), then use of Erythromycin in the unknown types of chicken bacteria will result in the development of a zone of inhibition if Gram-positive bacteria are present in the chicken bacteria?

Thank you Donna.

Hi,

Using pure cultures of S. epidermidis and E. coli would be excellent and would help validate your test method. Setting up the controls will involve quite a bit more work, but I think it would be worthwhile.

Do you have access to a microbiology laboratory to set up this experiment?


Donna Hardy

Good morning.

No, we don't have access to a lab. She has done an experiment with E.coli at home before and was able to get good results with no harmful consequences; though I did recently discover from reading other sections on your site that experiments involving bacteria are supposed to be done in a lab. However, I'm hoping she can go with this one since her procedure is due to be turned in early next week,and she hasn't been able to come up with any other ideas that she feels comfortable with/confident in.

She does know about proper sterile technique, wearing protective clothing, disinfecting work areas before and after, not opening plates once they have been inoculated, and proper disposal methods at the end of the experiment. Any suggestions, advice or warnings? You really have been so helpful, and we appreciate you.

Hi,

It sounds like you are taking proper precautions that will help avoid any problems in doing this project at home. After inoculation, you could tape the plates so there would be no possibility of accidentally opening them, although this will make measuring the zones of inhibition more difficult to measure. Take extra precautions if there will be any pets or young children around. Since you will be growing potentially pathogenic bacteria from raw chickens, make sure you have the teacher’s permission to do the project before proceeding.

What incubation temperature will be used for the experiment? The traditional temperature is 35 to 37 degrees Celsius. Also, what type of agar plates will you be using?


Donna Hardy

Hello.

Well, we don't have an incubator. We had read instructions on how to make one, but I was concerned that we could have problems with regulation of the temperature and that could negatively affect results. When Lauryn worked with E. coli cultures the one time before, she just put all of the plates on an out of the way shelf.. taped, agar side up in zippered plastic bags... at the same time. The shelf was not in direct sun nor was it near a heating vent; so I guess this wasn't the best method, but at least there was consistency, and all plates did grow a full lawn of bacteria, and zones of inhibition did develop. Of course, there are differences this time in using bacteria from chickens and antibiotic disks as opposed to paper disks wet from being dipped in green cleaners. Will this be okay? Other than taking longer for the growth to happen, does this method present problems?

As far as agar, I will be ordering prepared plates of nutrient agar from a scientific supply company.

Since your help, I am actually looking forward to her starting the experiment with my assist. I really enjoyed watching zones of inhibition develop the last time, and I am anxious to compare the results from the different types of chickens. We are totally open to any other ideas and suggestions you have, and I hope we will be able to ask more questions if we run into problems. But in the meantime, I just want to say thanks again to you and all the experts here. There is just so much information and good ideas on this site, it really is a wonderful resource!!

Hi,

You are absolutely right about temperature. The optimum temperature for bacterial growth is usually about 35-37 degrees Centigrade, but this is also close to the maximum temperature, usually 42-43, so it is easy to overheat the culture in an unregulated incubator. Many bacteria will grow well at ambient temperature and you will get consistent results with a constant temperature. Growth will be slower, so you might have to wait 48 hours to see the results. Temperature is one of the controlled parameters in this experiment, It would be a good idea to measure and record the temperature during the experiment.

Nutrient agar is a good all purpose bacterial medium, and will not support the growth of many pathogenic organisms, so is a much better choice compared to other media. However, Salmonella , which can be present on chickens, would be able to grow on nutrient agar at ambient temperature. Campylobacter, another group of pathogens that can be found on chickens, will not grow well on nutrient agar because it requires microaerophilic, or very low oxygen conditions. Order the plates right before you are going to use them, as plates can dry out quickly and dry plates will inhibit the growth of the bacteria. Since you have done a similar experiment before, you already know that the plates should be inverted after inoculation, and incubated upside down..

Please do let us know if you have any other questions. It sounds like you are well prepared for this experiment.


Donna Hardy

Hello everyone. Hope you are all having a good holiday season. Well, crunch time if finally here and I would really appreciate a bit more assistance. Lauryn has included 3 photos of the experiment.. there is one with a streptomycin disk on a plate of organic chicken bacteria. A zone of inhibition resulted with a few colonies in the zone. From reading, I understand that these colonies mean either there is some resistant bacteria, or more likely in my case, some different bacteria from the chicken, correct? Then there are two photos of a streptomycin disk on non-organic chicken bacteria...all five plates that were tested with a streptomycin disk on plates with non-organic chicken bacteria look like this. There is some clearing around the disk but it looks as if "tentacles" or "tendrils" are growing down towards the disk. I'm just not sure what is going on here. But maybe what is an even bigger problem.. or the cause of this problem... do I even have real lawns here? There is solid coverage over most of the plate, yet you can still see individual colonies spread throughout. What we did was..the chicken was swabbed and then that bacteria was then swabbed onto an agar plate and incubated at room temp. When colonies grew, a sterile swab was used to touch 5 colonies and then that bacteria was transferred into 10 ml of nutrient broth and left to incubate at room temperature overnight. Then two dilutions were done and the plates were inoculated. The agar plates then were incubated, again at room temperature, just shy of 48 hours. Is there an explanation for my bacterial "tendril" growth or do we just need to start again with better technique for a better lawn? As always, we appreciate your time and help.

Hi,

Thanks for posting the photographs. Your results are very exciting! It looks like you didn’t quite have a ‘lawn’ of bacteria. Ifyou had inoculated about twice as many organisms per plate, the growth would have been confluent. The plates are very consistent, however, so it looks like Lauryn had very good technique.

It looks like there was a partial inhibition of growth in the top two plates. However, you were working with a mixed culture and there was obviously at least one organism that was able to grow up to the edge of the plate, so your official reading on the top two plates is “zero mm. or no inhibition. The tentacle or tendril effect could be due to the motility of the organism. Was there any excess moisture on the two tendril plates? Confirming the reason for the tendril effect could be the subject of another very interesting science project.

After the overnight incubation at room temperature in 10 ml of nutrient broth, what were the two dilutions that were made before inoculation? Maybe just one dilution is needed. The right concentration of bacteria for a lawn is a suspension that is just barely turbid, or one that just has a slight haziness in the diluted culture.

The bottom plate shows a nice, clear, classic, and easy to measure zone of inhibition. Lauryn has shown a clear difference between the streptomycin resistance of the non-organic and organic chicken bacteria. If you have time, it would be great to do a repeat set of experiments with more than one brand of chicken, if possible. It would be a much more impressive project if the results are shown to be reproducible.


Donna Hardy

Hi Donna. Thanks for getting back to us.

No, I don't believe there was excess moisture on the non-organic chicken/streptomycin disk plates, and all five tested looked just like the photos above. As for the dilution, she used a sterile pipette to remove 1 ml of the 10 ml nutrient broth with chicken bacteria that had incubated over night and added it to 9 ml of sterile water and then used another pipette to move 1 ml of that tube to another tube containing 9 ml of sterile water.. so that is a 100 fold dilution? We will follow your advice and use just one dilution in another trial.

We were also interested to see that none of the penicillin g disks resulted in a zone of inhibition.. not on any of the 5 non-organic chicken plates nor on the 5 organic chicken plates. We considered that there was bacterial resistance to the penicillin disk, but then since Lauryn swabbed the internal cavity of the chicken.. and Gram-negative bacteria are often found in the intestines, we thought that it was more likely that the bacteria on the plates were Gram-negative. Does this seem reasonable? And I guess this whole question reveals a flaw in the experiment design.. it isn't exact or specific enough. For example, doing a Gram stain could have given us more information to work with. Any thoughts or advice are appreciated.

Hi Donna. If you are around, I'd like to get your input on this.. you told us that we should do two control plates to make sure our antibiotic disks were effective. Well, on the plate of E. coli, the streptomycin made a big, beautiful zone of inhibition while the penicillin G disk made no zone at all. Then on our plate of Gram positive bacteria, Micrococcus luteus, nothing happened.. nothing other than just a tiny bit of growth around the edges of the plates. We called the science supply company that we ordered from and was told that we should inoculate again and then put a cloth on top of the plate, letting it sit on our computer for warmth.. but still nothing. I remembered reading some of your answers before to people advising them to add a loop of the bacteria (though it was E.coli in these particular cases) to some broth and incubate overnight before inoculating their plates so that they would get a lawn due to the bacteria now being in the proper phase for good growth. Should this work for the Micrococcus luteus also? I would like that evidence that the disks are "working properly" especially since we haven't had any zones of inhibition produced from any of the penicillin G disks used on the organic or non-organic chickens.

As for the reasons for the no zones from the penicillin G disks, we are confused. We don't think it would be a bacterial resistance problem since the streptomycin disks made zones of inhibition on organic chicken bacteria {we did another trial after my post above this one and beautiful zones did result.. with no "tendril" growth this time.} So our train of thought was that Gram negative bacteria are associated with resistance problems moreso than Gram positive.. so if the streptomycin disks were able to make zones on the organic chicken, then we expected the penicillin disks to make even bigger zones. Especially since we were using organic chickens, which we didnt expect to have any resistance problems since they arent routinely fed any antibiotics , making resistance less an issue here than with the non-organic chickens. However, no zone growth resulted from the use of any penicillin G disk on either type chicken. Maybe this happened because we just happened by chance to select Gram negative bacteria from the plate of chicken bacteria that we grew in order to make our dilutions before inoculating for our "lawn plate"? Maybe because in slaughtering and processing, the intestinal bacterial from the chicken (mostly Gram-negative) contaminates the chicken? We are researching to find some answers. If you could offer us some insight and guidance on what is happening and/or where to look for further information, we would be very appreciative. Thanks.

Hi CJP,
Your penicillin control results with the E. coli are expected. Penicillin disrupts the cell wall of Gram-positive organisms, so you would not expect it to work against Gram-negative organisms like E. coli. The nice zone of inhibition you saw with the streptomycin disk confirms that this antibiotic disk was working properly.

The Micrococcus lutea did not grow at all so your control results for the penicillin disks could not be confirmed. You might want to politely request a refund for this culture from the scientific supply company, or if there’s time to repeat the penicillin control, ask them to rush a replacement.

Since you haven’t seen any zones of inhibition at all with the penicillin disks, all you can say is that either all of the bacteria in the samples are resistant to penicillin, or that the penicillin disks were not active. Gram-negative bacteria are very common in intestinal tract flora, and since they are motile, they might be overgrowing any Gram-positive bacteria in the samples.

You conducted the experiment properly with the appropriate controls, You can explain what happened and make conclusions based on your results. Since the penicillin control did not work, you can’t make conclusions about the penicillin results, but you can explain the science behind your project, and that’s important.

Donna Hardy