Best methodes for plating fratjuice

[dhimmel]

We recently froze some of the samples in preparation for 16S fratseq: see #2 (comment). However, we still have some refrigerated samples that we were thinking of plating. One goal would be to generate some nice Petri dish photos that help communicate the experimental design.

Does anyone have advice on what types of plate media we should use? Is a basic agar plate a good start?

[dhimmel]

Yesterday (2017-06-24), @bemert and I plated the samples on Lysogeny broth plates. I'll let @bemert fill in the details. Thanks to the following indivuduals for their plating advice:

Email from Elisabeth Bik on 2017-06-22

Elisabeth "Elies" Bik is a Science Editor at uBiome, who runs the popular Twitter account @MicrobiomDigest. She gave us the following advice:

For growing bacteria, of course with the restriction that many bacteria won't be able to grow, it would be best to pick a general rich nutrient medium, such as Luria Broth (LB) agar - without any antibiotics. For example, see this paper where environmental isolates from the ISS were grown.

LB agar is a very commonly used agar type. Prepoured plates can e.g be bought here or here

I would recommend making 10-fold serial dilutions of the fratjuice and plating out 100 ul per plate using a spreader or sterile glass beads (I can give more directions if needed). Some dilutions will produce confluent plates (too full), and others might not yield anything, but there will hopefully be a dilution/plate that will have the perfect amount of bacteria/fungi for a photo.

Email from Angelyca Jackson on 2017-06-23

Below is an email conversation with Angelyca Jackson where I asked her "Any advice on what media to use?" Angelyca is a scientist with the Deb Hogan Lab at Dartmouth. We met Angelyca when we gave the Hogan Lab a surprise visit the day we began the study. They set us on the right track and enlightened us a bit regarding microbes. Here's Angelyca's plating advice:

For plating the refrigerated samples I would plate on a couple of different non-selective media types for bacteria and for fungi. I recommend this because no matter what media you use, generally you will select for a certain type of 'bug' depending on the carbon, nitrogen, or other nutrient source within your plate.

Here are links to non-selective media for bacteria and for yeast and molds.

I would shoot for one that specifies that it's for broad-range culture without any inhibitors or indicators (so that you get the best chance of everything growing that's still alive in your samples). Keep in mind that your 16S fratseq (great name) data will be richer than what you may culture!

[bemert]

As Daniel mentioned, we plated each sample at two dilutions on LB agar. We diluted 10uL of each sample with 90uL LB broth then removed 10uL for a second dilution into 990uL LB broth. 90uL of the initial 1:10 dilution and 100uL of the 1 :1000 dilution were plated on LB agar and incubated overnight at 37ºC.
After incubating for 22 hours, all samples had colonies with the lesser dilution and only samples FJ2 and FJ3 had colonies at the higher dilution(1:100). As a non-microbiologist, I believe there are at least 4 distinct colony types in samples FJ1, FJ3, and FJ5, and at least 2 distinct colony types in samples FJ2 and FJ4. The smell reminds me of overgrown E. coli. Here are some images of the plates: