MAS.S66: How to Grow (Almost) Anything

Group 9


Diversity and the desires of individual humans microbiome

Our microbiomes are a complex set of living organisms within each of our bodies, attempting to grow and coexist within the human beings they inhabit. Too often, these organisms are subject to the whims of their host vessels, having their communities die and flourish based on the host's willingness to provide them with the resources and company to create a diverse space that everyone can grow in. Our microbiomes make up half of the cells in our bodies and control so many aspects of our lives. They deserve to be treated with respect. In our project, we will be looking at allowing the microbiomes of various people to interact with each other and to decide to communicate with minimal host input. This is an artistic and philosophical project as much as a scientific one-how do we treat and prioritize the living beings within us? How "human" are we really?

Flavor profiles

Motivating Questions:

At home microbiome sampling kits:

There are a bunch of apps to track food and nutrients. Sara tried out cronometer to see the breakdown of what she consumed.

Rae and Prathima developed a flovor profile survey to investigate what foods and flavors our team was particularly fond of. Below are the raw results:

Priya, Rae, Sara
Rahma, Yiqi

Lab notes:

May 10, 2019

After culturing each of our team members oral microbiomes on a large petri-dish...

We took a swipe off a colony and streached it on a plate to see and image the bacteria.
These are bacteria in Sara's spit. You can barely tell they're there with the 100x, the internet advises imaging with 400x-1000x to see their shape, but they were moving so we can tell these dots are alive.
A lot of measuring...

For each of the foods we chose, we prepared and autoclaved two mixtures:

  1. Media solution. Normally cells are cultured on 2.5% LB medium. We prepared food media, consisting of 1.25% LB and 1.25% food.
    Component Amount
    LB powder 1.25 grams
    Food (powder or solid) 1.25 grams
    Deionized water 100 mL
  2. Agar solution for plating. Agar plates are typically 2.5% LB and 1.5% agar. We prepared solutions of 1.25% LB, 1.25% food, and 1.5% agar.
    Component Amount
    LB powder 3.75 grams
    Food (powder or solid) 3.75 grams
    Agar 4.5 grams
    Deionized water 300 mL

May 11, 2019

Making the plates with different media

Today we planned to come in and plate the mixes we made yesterday, but unfortunately the autoclave was broken so we had to figure out another way to sterilize them. We ended up microwaving each flask for 3 minutes until the liquid boiled.

We had a lot of evaporation from the botched autoclave experience so the concentration of media in the flasks were much higher on the first plates. We didn't have enough liquid to plate all the pates we needed, so we ended up re-doing them all. Once the agar solutions were "autoclaved" in the microwave, we pipetted them into petri dishes to prepare agar plates of each food. The video below shows a few seconds of the production line plating 209 plates. 12 small plates and 7 large for each of the 11 media.

Liquid cultures

We also made liquid cultures for each media so we could measure the optical density as the bacteria grew or didn't grow as well. We took colonies from plates we'd cultured with out oral swabs and put them in LB. We also each spit into a tube of LB to see how the entire swatch of oral microbia would fair against the food.

May 12, 2019

Today we did three experiments:

Quantifying bacterial growth on the different food media

We restarted the overnight cultures by adding 50 uL of each culture to 5 mL of each food medium, and incubating for 3 hours.

Growing all our samples required a lot of labeling and some creating ways of fitting all the tubes onto a shaker in the warm room.

After the 3 hours, we measured took OD600 measurements of 200 uL of each sample in duplicate. We also placed the tubes back in the incubator to grow overnight.

Visualizing bacterial growth on the different food media

The previous day we'd tested the stamping procedure on one of the high-concentration-botched-autoclave plates and left it to incubate overnight. We found the colonies grew and stamping worked!

Therefore, on to production stamping, using a DIY replica plating method... we used a kimtech wrapped on one of the small petri dishes to do the stamping.

Capturing a view of the overall oral microbiome composition

Because we had cultured tubes with our spit, we thought it might be interesting to plate these samples to see if we got a more diverse picture of our oral microbiome. We pelleted 2 mL of the spit overnight culture in the centrifuged, resuspended in 400 uL LB, pipetted 50 uL onto each of our food agar plates (the ones with the smaller size), used glass beads to spread the bacteria, and incubated our plates.

May 13, 2019

We measured the OD600 of the bacteria samples that had been growing in the food solutions for about 20 hours in total, and checked on the growth on our plates.

Photoshoot and clean-up.

May 14, 2019

Bacterial imaging

We tried again to image some bacterial samples, this time with a higher optical objective and also with an SEM. We took a colony from an agar plate and wiped it onto a gold coated silicon wafer which allows the electrons to refelect back. The first picture below is the machine uses. The middle picture is the prepped sample, and the picture on the far right is the sample going into the machine.

We couldn't see much with the SEM, only the surface of the sample. There were a lot of cracks and misleading details in the sample, which could have been bacteria, or could have been dust or bubbles. At this scale, everything looks amorphous. The concentration of bacteria seems too low to get a good image, and we learned there are a number of protocols followed to get the amazing SEM images seen in text books, mainly fixing a suspension and staining the cells. This is an active area of research, and a topic people are still writing papers on.

Back at the good old optical microscope we put an entire petri dish under the objective. It was really hard to ajust, because the surface of the culture wasn't a uniform height, but we did get a couple images seen below. The left image is at 100x and the right is at 200x. This is still a smaller magnification than we'd need to see a 1-10um thing, i.e. bacteria, and it's unclear what these images are actually of.

Sample preservation

To preserve the plated cultures Rae potted them in epoxy.


Concluding slides

Future work

Making a bioprinter with gradients: print out a sculpture with a food gradient designed such that, when we add the microbiomes of two people, we can accurately predict where each person's microbiome will dominate.

Materials used