Science Buddies: "Ask an Expert"

For my project, The effect of unsaturated fatty acids on E.coli and spinach, I don't know what to make my media out of. Someone please tell me with what can I make my media with!

For my project, The effect of unsaturated fatty acids on E.coli and spinach, I don't know what to make my media out of. Someone please tell me with what can I make my media with! Also, what can I measure, after I have cultured the bacteria??? I don't want to measure the Zone of Inhibition, but anything else I am willing to do.. so any suggestions.

Hello there,

First of all, would you mind giving a brief description of what your project is about, what question(s) you're trying to test, and what your hypotheses are? It would be really great so I can give you as much helpful advice as possible! For example, are you throwing some unsaturated fatty acids on E. coli and spinach and seeing what happens to them? What will your read outs be though? In other words, what would you expect to see? Would you expect the unsaturated fatty acids to help E. coli grow better, or kill E. coli? What about the spinach? Why are you comparing the effects of unsaturated fatty acids on a bacterium versus a vegetable? Why not compare the effects of the unsaturated fatty acids on two different strains of bacteria, or two different types of vegetables? Where are you going to get these "unsaturated fatty acids"?

When you refer to media, are you referring to the media that you're going to culture your bacteria in? If you want to grow E. coli, you should probably use Lb. A very common recipe for preparing 1 L of Lb media 10g tryptone, 5g yeast extract, and 10g NaCl, and bring your mixture up to 1 L with distilled water. However, there is also Lb powder that you can purchase, and you can just suspend whatever amount the vendors recommend with a certain volume of distilled water. You can then grow E. coli in this liquid media! Furthermore, you can culture your bacteria on Lb agar plates, where you can just add some agar to your Lb media and pour the mixture into plates and let it solidify. Then, you can streak out your bacteria onto those plates. Do you have all the materials and resources to make this media and the plates though? Are you working with a mentor or teacher who can give you some guidance as you make these things?

As for what you can measure after you culture the bacteria, I'm having a little trouble understanding what you want to do for your experiment. Are you going to culture your bacteria in the presence and absence of unsaturated fatty acids, and then compare what happens when you add the fatty acids to the bacteria? One thing you can do is generate a growth curve for the bacteria grown with or without the fatty acids. At different time points, you can take a sample from your liquid culture and plate it to count the number of bacteria present in your culture, and then plot bacterial numbers versus time. There, you can compare how the bacteria grow when you give them unsaturated fatty acids versus when you do not. If you want to learn more about how to do this, let me know and I'll give you more details! What will you do with the spinach though? Also, have you done some research on how unsaturated fatty acids affect E. coli? This may give you some more clues on what you can test!

Anyway, hope this helps. If you tell me a little more about what you are trying to do with your project, I can give you more insight as to what types of experiments you can perform and what types of observations you can make!

Best of luck,

Connie

Hello there,

Please refer to my reply in the other thread you posted ("The effect of unsaturated fatty acids on E.coli and spinch") about how to make your media!

Best,
Connie

Hi,
What I am going to do is have the agar plates and have the E.coli and spinach on the plates and let the bacteria culture to try to make a new strain. Then I am going to add unsaturated fatty acids and see the results to see if the inhibition of bacteria is less. The obstacle that I am in now is that I don't know what to measure, because I do not want to measure the zone of inhibition because many people do that. I was originally going to measure the thickness of the biofilms, but I would have to go to a university to get accurate results, and even then, I could not test all of my petri dishes.So, I want to know what I should look for. The reason why I am using spinach is because many outbreaks have been seen to show that leafy vegetables, such as spinach and lettuce, inhibit bacteria. I want to make an E.coli strain that is closest to E.coli 0157 to use for my project, so that I would get more accurate results.
Thanks!

Hello there,

I am still slightly confused as to the main objective of your project. Let’s try to work this out together.

1. First, you mention that you are going to put E. coli and spinach on agar plates, and culture the bacteria in order to generate a new strain, preferably something that is closest to E. coli O157. I am curious as to why you think putting E. coli on spinach will allow you to generate a strain that is like O157. Indeed, E. coli O157 outbreaks have been attributed to spinach contaminated with the bacterium, but that does not necessarily mean that the spinach is responsible for generating O157 as a strain. Bacterial interactions with various environmental and host factors likely contributed to the generation of the O157 strain, and culturing normal E. coli on spinach alone will very likely not cause the exact same mutations and adaptations that have generated the O157 strain. For example, one feature that distinguishes normal, harmless E. coli from pathogenic E. coli O157 is that O157 produces certain toxins that harmless E. coli doesn’t—culturing normal E. coli on spinach will not cause the bacterium to suddenly acquire the ability to make those toxins. Furthermore, if you were to generate an E. coli strain that is close to O157, you will have to worry about biosafety. You cannot work with a potential pathogenic strain in your own home or in the classroom—you need to work in a lab with the proper equipment and biosafety certification. I’m not sure if I understood what you are trying to do with the spinach correctly, so if what I just mentioned is not what you had intended to do, let me know!

2. You mention that spinach and lettuce has been shown in certain outbreaks to inhibit bacteria. I’ve heard lots of outbreaks associated with contamination of leafy vegetables with bacteria, but not many outbreaks associated with the leafy vegetables actually INHIBITING the growth of the bacteria. However, I did find some research articles that mention that oils produced by plants and vegetables actually do have antimicrobial activity. I didn’t find much about spinach having antimicrobial activity, but I did find some stuff on basil, thyme, etc. If you have the primary source that claims that spinach has antimicrobial activity, I would be curious to take a look at that. Also… you had just mentioned that you want to generate a new strain of E. coli by growing it with spinach, and now you’re mentioning that you think leafy greens inhibit bacterial growth. Let’s say that leafy greens do inhibit bacterial growth… then wouldn’t culturing E. coli on the spinach be contradictory? Wouldn’t you not even get any bacteria growing if you were to culture it with something that is supposedly “antibacterial”?

3. Finally, you mention that you want to add unsaturated fatty acids to see if the inhibition is less. What are you adding the unsaturated fatty acids to? The E. coli cultured with spinach? In that case, why do you think that the unsaturated fatty acids will actually lessen the inhibition? Have you done some reading and research and found something about unsaturated fatty acids making antimicrobials less effective? If you did find something like that, again I would be curious to take a look at it so I can have a better understanding of your reasoning for doing this. Unsaturated fatty acids have actually been shown to have antimicrobial activity, so I am curious as to why you think that unsaturated fatty acids will “lessen the inhibition”.

4. Instead of measuring the zone of inhibition or biofilm thickness, you can just measure bacterial numbers overtime. You can treat your bacterial culture with whatever unsaturated fatty acid you had intended, and at different time points, count the number of bacteria present in your culture. You should have an untreated culture as your control—you should see that the E. coli are growing and hence their bacterial numbers should increase overtime. However, if you add some sort of antimicrobial to the culture, you may then notice that the bacterial numbers will gradually decrease overtime because the bacteria are dying. Let me know if you want more details for how to do this—this is a really standard way of monitoring bacterial growth and the effects of antimicrobials on the bacterium, and is also very simple to do!

Anyway, I hope all of these questions can help you re-evaluate your project and see whether there are certain aspects that need fixing. I hope I did not misunderstand your explanation of your project ideas, but please write back with more questions and comments, and I will try my best to help you.

Best,
Connie

Hi,
These are a few articles that I have cited from my introduction,

Furukawa, S., Akiyoshi, Y., O’Toole, G. A., Ogihara, H., & Morinaga, Y. (2010, January 7).
Sugar fatty acid esters inhibit biofilm formation by food-borne pathogenic bacteria. Pub
Med Central. doi: 10.1016/j.ijfoodmicro.2009.12.026

Kyle, L. Jennifer, Parker, T. Craig, Goudeau, Danielle, Brandi, T. Maria. (2010). Transcriptome
analysis of Escherichia coli O157:H7 exposed to lysates of lettuce leaves. Applied and
Environmental Microbiology. Retrieved from http://aem.asm.org/content/76/5/1375.full

Carter, Q. Michelle, Xiue, Kai, Brandi, T. Maria, Liu, Feifei, Wu, Liyou, Loiue, W.
Jacqueline, Mandrell, E. Rbert, Zhou, Jizhong. (2012) Functional metagenomic of
escherichia coli 0157:H7 interactions with spinach indigenous microorganisms during
biofilm formation. PLOS ONE. Retrieved from:

I am trying to put the omega family unsaturated fatty acids in the petri dishes and trying to see if the growth of the pathogen decreases. The purpose of this project is to lessen the pathogens on processed foods, so I am trying to see if these acids would do.

Hello there,

Those articles made things so much clearer! Now, if I understand correctly, what you want to do is to grow E. coli on spinach (this is to mimic E. coli O157 contamination of leafy vegetables that were responsible for several terrible outbreaks), and add omega family unsaturated fatty acids to see if that would inhibit the growth of E. coli on the spinach. That makes a lot more sense to me now, and so now I think that I can give you more specific advice on what you can do. This is a very interesting and relevant topic, considering how food contamination with microbes has huge impacts on the human population as well as the food industry. Great job coming up with this idea!

First of all, I would suggest you to just use normal non-pathogenic E. coli as a model as opposed to actually trying to generate an E. coli O157-like strain because first of all, it’ll be nearly impossible to do that unless you have the proper lab equipment, and second of all, because O157 is pathogenic, you cannot work on this in your own home and you need biosafety certification. Non-pathogenic E. coli will work well enough as a model for O157. Make sure you check out the Microorganism Safety Guide on Science Buddies to ensure that you are working as safely as possible with these bacteria! https://www.sciencebuddies.org/science- ... fety.shtml

Second of all, as to how to grow the E. coli on the spinach, I would probably follow the protocol from the third paper you sent me (Functional metagenomic of Escherichia coli O157:H7 interactions with spinach indigenous microorganisms during biofilm formation). For normal maintenance of E. coli and to grow up enough bacteria to use for your experiments, I would first grow them in Lb liquid media or Lb agar plates. Do you have any access to a university lab or a centrifuge? If you do, that would be very helpful in preparing your cultures, the Lb media and/or plates, and the spinach leaf lysates. For your experiments, I would follow the methods in that third paper: I would prepare the fresh spinach leaf lysates as they mention. Homogenize the same amount of spinach leaves and resuspend that in the same amount of water if necessary in order to keep a constant concentration for all the cultures that you are going to set up. I would probably set up two different liquid cultures in bacterial culture tubes (like so: http://www.ridge2000.org/seas/for_stude ... -tubes.jpg): (1) E. coli + spinach leaf lysate + buffer, (2) E. coli + spinach leaf lysate + fatty acids resuspended in buffer. After preparing the spinach leaf lysates, I would then take an overnight culture of E. coli in Lb liquid media, spin that down using a centrifuge, and resuspend the bacteria in some saline solution (they used 0.85% NaCl). I would then put in the same volume of bacteria into each culture tube that you have set up with the spinach leaf lysate. However many bacteria you want to put in depends on you, but the paper started at about 10^4 bacteria/mL. Finally, I would resuspend the fatty acids in whatever buffer that the vendor suggests. One thing to remember, however, is that the longer the carbon chains get on your fatty acids, the more hydrophobic it becomes, and it may be difficult to get them to solubilize in your E. coli spinach leaf lysate culture. Then, I would add the fatty acids to one of your E. coli spinach cultures, and just plain resuspension buffer into the other culture as a negative control.

Since you want to determine whether the fatty acids can inhibit bacterial growth, the easiest thing to do is to generate a growth curve for your two cultures, where you will monitor the bacterial numbers at different time points starting at Time 0 over the course of their growth. However many time points you take really depends on you, but it seems like for the third paper you sent me, they monitored the growth of the E. coli in spinach lysates for about 2 days (Figure 1)—hence, I would probably monitor the growth over the course of 2 days, and perhaps take samples from your cultures at Time 0, 12h, 24h, and 48h and count the bacterial numbers at those times. You can count bacteria via a method called serial dilution, and there is a link here explaining how to do so: http://www.disknet.com/indiana_biolab/b038.htm. If the page doesn’t explain the procedure too clearly, feel free to shoot me another comment and I can explain it to you. In the bacterial culture that has E. coli, spinach lysate, and only resuspension buffer, you would expect the bacterial numbers to increase overtime, since you don’t have the “inhibitory” fatty acids in them. As for the culture with the fatty acids, that is up to you to find out through your experiments whether the compounds do inhibit bacterial growth!

In addition to doing the growth curve, you can also measure the thickness of the biofilms as you mentioned before if you have the resources to do so, since lots of foodborne pathogens do form biofilms on the surfaces of the food to which they’re attached. This will also give your project another dimension: Not only have you monitored the effects of fatty acids on the growth of the bacteria on spinach lysates, but also on how it affects biofilm formation.

I hope this was helpful. Feel free to send us anymore questions if there is any confusion.

Best of luck,
Connie

I am going to do 120 petri dishes in total, so would doing serial dilution be efficient? Could i not just count them at 0, 12, 24,and 48 hours?

You're going to do 120 petri dishes? Why so many? Are they all replicates of each other, or are you testing a whole bunch of compounds? Also, if you are going to grow bacterial cultures and count them, it would be easier to do them in liquid media in bacterial culture tubes. The serial dilution is necessary for the counting, because think about it. If the bacterial numbers get up to 10^8 bacteria per ml, are you going to count 10^8 bacteria for all 120 of your samples? The point of taking a small sample from each of your cultures and serially diluting it is so you can dilute the bacterial numbers to levels that can be reasonably counted (for example, 10-100 bacteria as opposed to 10^8). Then, you can back-calculate from the dilution you've counted in order to determine what the original concentration of your culture was at that certain timepoint. Here is a video on serial dilution that explains the technique and the reasoning behind using this to count bacterial numbers: http://www.youtube.com/watch?v=pmRUBYlPMBM.

Our teacher said that he wanted us to have 30 trials for each independent level for a good amount of replication, because many judges ask if we have done replication. So that is why I am doing 120.

Hi Sciencegeek,

Connie seems like she has really been steering you well so far, and I just wanted to enforce her recommendation to use serial dilutions. As someone who has spent A LOT of time counting bacterial colonies on plates, it will be much less painful for you if you have ~100 colonies per plate, and since you'll have no way to know how many bacteria are in your liquid cultures, the only way to ensure that you have a countable number of colonies is to do serial dilutions. Take my advice, you do not want to be attempting to count 1,000 colonies on each plate (not to mention that this will make your final counts much less accurate).

JMP

Ok thanks! I will use this in my methods! Thanks for both of yall's help!!