Pseudomonas aeruginosa Biofilm Foldscope Exploration

Hi Everyone,

In my first post, I talked a little bit about my microbiology independent research project that I have been working on for several months now. My project’s focus was destroying P. aeruginosa biofilm formations with isolated bacteriophages.

My original method (the one talked about in my first post) was to grow different triple culture combinations of P. aeruginosa, Staphylococcus aureus, and Escherichia coli overnight and then plate them on a P. aeruginosa lawn. When I did this, I observed zones of clearance around the areas where I plated the triple cultures on the lawn. However, when I took samples from those areas, grew them overnight in nutrient broth, centrifuged, filtered, and plated them on a P. aeruginosa lawn, no zones of clearance were formed as shown by the picture below. I only noticed plaques formed on top of the lawn. This is an interesting result in itself as it shows that whatever is occurring with triple cultures is giving the bacteria the ability to successfully establish a colony on the P. aeruginosa lawn whereas if E. coli or S. aureus were placed on a P. aeruginosa lawn by themselves, they would be overrun by the lawn.

This was not enough. I wanted to create a plaqueless zone of clearance on the lawn, which is a clear indicator of a toxin or bacteriophage. I did some research and found an interesting procedure that described how to isolate bacteriophages that target P. aeruginosa from a solid sample. The solid samples I used were soil samples from three different locations in my school’s garden: a plant bed, the compost heap, and a dry soil heap. I took 1.5 grams of a soil type and treated it with SM Buffer. I took about 1.5 mL of the liquid soil/SM Buffer solution and centrifuged it for 10 minutes and filtered the supernatant  with a 0.45 micrometer filter. I then took about 1 mL of this solution and mixed it with 8.5 mL of nutrient broth and 0.5 mL of an overnight P. aeruginosa bacterial culture and grew it for 24 hours. After 24 hours, I divided each of these cultures in multiple Eppendorf tubes and centrifuged them. I filtered the supernatant and placed it on a P. aeruginosa lawn and incubated the plate for 24 hours. This process was done for each soil type.

I did this experimental process twice and I got very interesting results both times. The 2nd time I did the experiment, I clearly noticed a plaqueless zone of inhibition in the lower left quadrant where I plated the refined sample from the dry soil, which is an indicator for a bacteriophage or toxin effective against P. aeruginosa as a competing bacteria would establish a colony in the middle of the zone of inhibition. The large zone of clearance in the bottom right corner is the result of a positive control, Ciprofloxacin. Check it out right here.

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In my first post, someone gave me a great suggestion in the comments and said that I should try to observe biofilms with the Foldscope, so now that I have a better idea of how to use the Foldscope, I decided to give it a shot. I took a sample from the area near the zone of clearance formed on the 2nd plate, made a microscope slide with it, and began exploring! I also tried making observations with my school’s microscope and took photos from both explorations. The 1st photo was taken from the school microscope and the 2nd was taken from the Foldscope.

 

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It honestly boggles my mind how a microscope that costs roughly $1 to make provides the observer with a better quality image than a traditional microscope and is more accessible as taking photos and panning around images is significantly easier. On the traditional microscope, you get a clear overall view of the sample, but the Foldscope lets me see that and the smaller details. The small “grains” visible embedded in the purple  areas that represent the extracellular biofilm matrix are the P. aeruginosa bacterial cells.

More Foldscope Images

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I am really really impressed by how amazing, practical, and minimalistic the Foldscope is. Absolutely amazing!

Thank you everyone,

Leonardo Biral

6 Comments Add yours

  1. Manu Prakash says:

    @ Leonardo; that is an incredible exploration. Specially since community growth and development in microbial form is such a crucial but relatively unexplored area. Just a week ago, a faculty from MIT (and a great friend) talked about bulk culture experiments of growing groups of three bacteria found in soil together. They have found some very puzzling results (not obvious if its possible to predict what grows with what). So you have a fantastic series of experiments ahead.

    Also; here are some more tips on illumination that will give you even better (and flat) field of view.
    https://microcosmos.foldscope.com/?p=9289

    Also; try to setup focus locking using this simple trick – you will be thrilled you read this post (unless you already have)
    https://microcosmos.foldscope.com/?p=16093&

    I am inspired by your explorations..

    cheers
    manu

    1. Saad Bhamla says:

      Awesome post Leonardo. Really great to see your experiments coming along well and your images are quite beautiful.

      Thanks for sharing and looking forward to your next posts:)

      Saad

      1. leo10bi says:

        Thank you very much Saad. I really appreciate the kind words.

        – Leo

    2. leo10bi says:

      Thank you for sharing your very helpful posts with me Manu. I definitely will start using the tips you give in your posts in future explorations.

      Bacterial community growth is an extremely interesting field of study. In my experience, when P. aeruginosa, S. aureus, and E. coli triple cultures are grown together and then plated on a P. aeruginosa lawn, plaques with zones of clearance are formed. However, similar plaques do not form on the P. aeruginosa when double cultures of any two of those bacteria are grown together overnight and then plated. There is something special that occurs when those 3 bacteria are combined that is really worth pursuing.

      I am very curious to know what your friend at MIT found in his experiments with triple cultures. It is a very fascinating, deep research field that I definitely will want to pursue during the summer (if I can find a lab to continue working at), Senior year, and later at university !

  2. Manu Prakash says:

    Dear @Leo,

    Here is the series of papers I meant; the one I was referring to is not published as yet. But you can see this: http://www.pnas.org/content/113/22/6236.full
    and this: http://www.gorelab.org/publications.html

    cheers
    manu

    1. leo10bi says:

      Thank you so much for those very interesting articles Manu. The findings of the 1st article are especially significant as they show that bacterial mutualism between different bacterial strains is a common occurence that plays a significant role in the growing problem of antibiotic resistance. It is amazing how these bacteria can sense that a different bacterial strain can help them survive and they work together to survive. This could very be what happened during my experiment with S. aureus and E. coli being able to successfully compete against the P. aeruginosa.

      I will definitely show this article to my project mentor and keep these findings in mind during future experimentation.

      Thanks again Manu!

      – Leonardo Biral

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