Science Buddies: "Ask an Expert"

Hello, I really need some help on my project! I'm planning on observing bacteria in yogurt on a microscope. Would a good experiment be to test if leaving yogurt out for for a certain amount of time (x days) in warm temperatures affect population of bacteria? If not, then do you have any other recommendations I can test that still has to do with yogurt bacteria? Thanks!

Hi,

It is actually quite difficult to observe bacteria in a microscope. It requires some special fairly specialized microscopes, so unless you are sure you have that available to you, I would recommend buying agar plates and plan to plate your different yogurt samples to count the number of bacteria present. As for ways to change up your project some, I recommend that you search our Science Fair project ideas here. I did a quick search for yogurt and bacteria and found three projects listed:
https://www.sciencebuddies.org/science- ... ml#summary
https://www.sciencebuddies.org/science- ... p010.shtml
https://www.sciencebuddies.org/science- ... p072.shtml

Maybe one of those will appeal to you, or you can figure out how to tweak those to make them more your own project.

Hope this helps and please feel free to keep posting in this forum if you run into any more issues.

JMP

Oh my goodness! Thank you so much! This helped lots. I am excited for this project. I'll use agar plates for plaque assays!

Hello there,

JMP found some very interesting project ideas for you! Let us know if there are certain tweaks you want to make to make the project your own, and if you need any help with brainstorming.

Just out of curiosity, why are you thinking of using agar plates for "plaque assays"? As far as I know, people don't ever really use plaque assays for bacteria--they only do for viruses. Or are you thinking about some other type of assay?

Let us know if you need more help!

Best,
Connie

Well, there are bacterial assays, according to some research I have done. I guess I don't really know the name of what I want to do, but I do know that I will use the agar plates to count the colonies of bacteria that grow. Is there a specific name for that? Bacterial assay? Thanks!

~VirusDrM

Oh, and I forgot to ask, how do you tell if there are different types of bacteria colonizing in the agar plates? Are they different colors or something? I was also thinking of diluting the yogurt in order to make the colonies easier to count. When counting the colonies, does it matter if they're of the same genus and species? Or can I just count the bacterial colonies REGARDLESS of the type? I need to know this for when I graph the bacterial curve. Thanks for your time in answering these questions!

~VirusDrM

Hello there,

Ah, I believe the assay you're talking about is just plating bacterial colonies onto agar plates and counting for CFUs (colony forming units). I know that you have read up on the methods for doing this, but let us know if you need anymore help with the procedures!

Honestly, if you want to tell if there are different types of bacteria colonizing the plates, it really depends. For example, different bacteria may form colonies of different sizes or colors, and that way you can tell them apart. However, there are also different species of bacteria that can form very similar looking colonies. In that case, you would need more sophisticated molecular techniques to tell how many species and what different species are on your plates. If you know from the start what bacterial strains are in your yogurt, you can google the species and see what colonies of these bacteria may look like, and use that as your reference. What are you interested in analyzing specifically? Do you want to see if there are different bacterial strains between different types of yogurt?

Feel free to post back with anymore questions you may have. Hope this helped!

Best,
Connie

Thanks for the info! It's super helpful! What I'm trying to analyze is if the time of incubation of the yogurt affects the population of the bacteria. However, what really confuses me is that there are different species within the yogurt. I do not know how to graph the CFUs for that. I also do not know if I should count ALL the colonies REGARDLESS of the bacterial species and graph the CFU/mL of that. Do I need to culture streak for each species and analyze for each or just count all the colonies despite the mixed species in the agar plate?

For my experiment, I was thinking of doing 7 trials for 3 groups: under incubated, well incubated, and over incubated yogurt. That would mean 21 agar plates. One problem. If I dilute each sample five times, then I would need 105 agar plates! Too many to fit in an incubator!! Even diluting them 3 times gets me a high number of agar plates! What can I do to make the number of agar plates more manageable while still having reliable data (countable colonies)? Sorry for throwing so many questions at you all at once, but I really want thorough and accurate data. THANKS FOR YOUR TIME!

~VirusDrM

Hey,

For counting the colonies, to keep it simple, I would just count all the colonies despite the mixed species in the agar plates, if your main question is to see whether there is a change in bacterial numbers depending on the incubation conditions of the yogurt. However, if you want to see whether different incubation conditions affect individual bacterial species within yogurt, then you would need more sophisticated molecular techniques to address that question. It would likely require you to find a local lab to help you with those experiments.

105 agar plates is a lot of plates! I was wondering why you decided to do 7 trials for your three groups? Many scientists tend to do triplicates for their experiments (so only three trials). If there is no specific reason why you need to do 7 trials and you can lower it down to 3 trials instead, that can bring you down to 45 plates if you do five dilutions for each, which is definitely doable.

Your experiments seem to require methods that are pretty well explained in a link that JMP had posted to you earlier on a project called "Is That Really Bacteria Living In My Yogurt?" (https://www.sciencebuddies.org/science- ... ml#summary). You should definitely check the background and procedures sections out. One very important thing I found from this page was that they suggested growing your plates in an anaerobic chamber (no oxygen) because many of the bacterial species put into yogurt naturally live in anaerobic conditions.

Hope that helped!

Best,
Connie

Excellent! Thank you for the info. The reason why I wanted to do 7 trials was because I initially suggested 5 to my science teacher; but he said more than 5. I'll tell him about the scientists doing triplicates so that he won't have to order so many agar plates. Thanks for helping me make this experiment more manageable.

Since I will be making my own yogurt, will it be okay to incubate the yogurt in the agar plates? What I was thinking of doing is to have a helper help me dilute the yogurt (not yet incubated) so that the inevitable lab error of not putting all plates in the incubator at the same time, is violated too drastically. Then I would put them in the incubator as soon as agar plates are done, then keep on diluting until all are done. Or can PRE-MADE yogurt be incubated? And do you think I should do all 3 trials in one day or expand it to 1 trial per day with a total of 3 days? If my plan does not seem good enough, suggestions would be nice. PLEASE ANSWER and thanks for your time!

~VirusDrM

Hi,

Okay, I was thinking of everything I would need to do at school to do this project and I found that I probably won't be able to culture my own yogurt, so I was reading up that yogurt bacteria helps with halitosis by killing bad breath bacteria in the mouth. I want to change my experiment to address that by swabbing my mouth and growing my mouth bacteria in petri dishes, incubating them, then applying some sugar-free yogurt (Nancy's Plain Yogurt and Activia Plain Yogurt) on the bad bacteria in the agar plates. Then incubate that and see which brand of yogurt bacteria kills or impedes the growth of bad mouth bacteria best. One thing, how would I dilute my saliva to get countable colonies? Take 1mL of it and dilute it regularly? Is my plan good? If you have any suggestions for improvement or good websites that I probably did not look up, please post them!

I also feel rushed since I have this week and Sunday to put my FINAL plan together and present it to my science teacher, who wants it ready by Monday. Please answer as soon as you can! Thanks!

~VirusDrM

Hi,

I think you've come up with a really interesting project, and I'm glad you're so interested in this, because the best projects are always driven by your own interests. As for diluting saliva to plate for bacteria, I have to tell you that I don't know how many bacterial colonies you'll get out of your saliva, so I can't tell you how much to dilute it. In the lab, we would start by doing a little "pre-test" and plating some serial dilutions of saliva so we can determine how much to plate in future experiments. Basically, I would take some of your saliva and plate a set amount of it directly, without dilution. Then you can take the rest of your saliva and dilute it two-fold in water (preferably sterile water). Plate half of that, and dilute the rest two-fold in water again, and you just keep doing this until you have done a whole series of dilutions. Then you let that grow and see which dilution gives you a good, countable number of bacteria, and that is how you would dilute the saliva for your experiment with the yogurt. If I had to guess, I would think that you will not have to dilute it very much, if at all, to get countable colonies, but I could be very wrong.

As for adding yogurt to the plates and seeing if they kill or impede the growth of the "bad" bacteria, my biggest concern is that it will be difficult for you to tell the difference between the "bad" bacteria and the "good" bacteria in the yogurt that may also grow on the agar. As we said, identifying different bacterial strains can be quite difficult without sophisticated techniques and lots of experience. Can you think of any controls you could include to help you with this problem? Think about it, and post again if you need help there, or make any other changes.

JMP

Hey thanks! I was concerned about telling the difference between the good and bad bacteria too. I think that the pretest would help a lot. Perhaps I could take pictures of the pretest agar plates so that I won't forget what the "bad" colonies look like. Then, once the good bacteria grow on it, I might be able to tell the difference between the good and bad bacteria. I will also do some research for that.

~VirusDrM

Hey there,

JMP gave you some really great advice, and I think your new question is very interesting. I would add that in addition to plating the bacteria from your mouth as a pre-test, you should do the same thing for the bacteria in the yogurt just to see how the "good" colonies would look like. My biggest concern though is that sometimes, different strains of bacteria may have similar colony morphologies, which would make it very difficult for you to tell the difference between the good and bad bacteria. In this case, you may need more sophisticated molecular techniques to tell the difference, and this would depend on what kind of resources are available to you in your high school lab. You may consider discussing your new idea with your teacher and see if you guys can come up with an experiment to test your question with the materials that you have in your lab.

Feel free to post back with more questions! We'd be happy to brainstorm with you.

Best,
Connie

Hello!

I like that extra suggestion. Yes, I will definitely mention that to my science teacher. Hmm, that would mean I need 18 vials for the pre-test on both oral and yogurt bacterial dilutions. I will be doing a triplicate for the oral bacteria, then just one pre-trial for each brand of yogurt since it will be for just observing the look of the "good" colony.

~VirusDrM

Hi, will you please help me out? I did my pre-experiment and put 18 agar plates in an incubator at 38 degrees Celsius. They have been in the incubator since Thursday of last week and are not growing! I pipetted 1 mL of saliva from a scale of not diluted to a 10-5 dilution. How can I get them to grow faster? The science fair is on March 7, and I have not done that actual experiment yet! Please answer ASAP.
Plus, I put the two agar plates with Nancy's Plain Nonfat Yogurt and Activia yogurt.

I appreciate the help!

~VirusDrM

Hi - I am joining this discussion after reading all the interesting posts that led to your really interesting experiment! I'm very interested in what you will discover because in my personal life I've seen much improved periodontal/gum health over the past years that yogurt has been an almost daily part of my diet.
As a high school biology teacher I have watched students plate what we knew were live stock colonies and come up with no growth. When I watched the students' technique for plating they simply did not wait for the inoculating loop to cool enough before dipping into the colonies to be plated. So, they had killed them and were plating dead bacteria which of course would not grow. So, I wonder if you followed a step that will avoid this - that is to touch the hot inoculating loop to the edge of the agar to cool the loop, then use the loop to catch and spread your bacteria.
Just a thought.

Good luck! I hope you can get this going - I'd love to find out I'm eating yogurt for more then my digestive system!

Yvette

Thanks for the advice, however I am using rubbing alcohol to sanitize the makeshift loop (paper clip rounded at the end for the "loop") and dry it off to get the rubbing alcohol off. Is it the temperature that's wrong? Should I inoculate it differently? Or is the fact that I stored my saliva in a capped graduated cylinder overnight and then used it after school to plate killed the bacteria? Please help!

Plus, can CFUs be calculated without dilutions and just counting the colonies? I thought of a backup plan to save time and want to swab my mouth and inoculate each plate that way without diluting.

Oh - then you are not killing the bacteria with as I thought might have happened.

Is it something that was in the graduated cylinder? Could there have been some soap left in it or whatever you used to clean it with?
I guess I would try again as soon as possible.

Also, I know the paper clip loop is used in the procedure for this experiment, but in class we've had good luck with students collecting and streaking plates using the sterile packaged throat culture swabs that drs use to collect bacteria. Works well and students don't have problems with stabbing the agar too hard. Of course use a new one for each inoculation and open the paper they are packaged in on the long stick side, keeping the cotton swab covered until ready to collect the bacteria.

I'm wondering also if the saliva simply doesn't 'stick' enough to the smooth paper clip and essentially not enough bacteria can be transferred from his collection solution. The Q-tip swab drs use for throat cultures would soak up his saliva solution and he can gently but firmly spread over the agar plates.

Other experts - is this acceptable for this experiment?

Good luck!

Another question. Since I am growing saliva bacteria, do I need to incubate them
at 38 degrees Celsius or can I just leave them at room temperature?

Hi -
Bacteria that you find in your body is growing at body temp (98.6F or 37C); If you leave the bacteria at room temperature it will most likely be anywhere between 68 - 78 F (25 degrees C)- (depending where you live if the heat or A/C is on this time of year?) - so, you see this bacteria left at room temp will grow slower than if you incubate them at 38C which is about your body temperature.

You have helped me so much! My experiment is saved.Thanks. Also, I did not realize that the agar was supposed to absorb the saliva. I left it right on top and liquid. I did not spread it well enough to absorb. I did so much research now I am confident the experiment will turn out well.

Glad to be able to offer some tips along with all the excellent support you've gotten from all the other experts here.
Now you are learning that doing science is really all about trial and error and constantly fine tuning techniques and procedures. Read as much as you can about the set up and techniques - and always leave enough time for the first or even the second try to need improvements.
Keep us posted on how things are going.