Sterilized Tryptic Soy Broth (TSB) sits on the microbiology lab bench, ready to be used in the growth of experimental cultures. The broth will be inoculated from a starter culture of B. subtilis. Cultures are grown in liquid medium and on solid medium as well, at 37º C for 48 hours; liquid cultures are shaken at ~225 rpm.

Trytpic Soy Agar (TSA) plates wait on the lab bench to be bagged. Plates are often poured in advance and stored in a cold room until needed. To prevent plates from drying out, they are stored in sealed plastic bags.

Lesley prepares to streak a starter culture of B. subtilis from a stock culture. Starter cultures are streaked onto plates made with Schaeffer’s Sporulation Agar (SSA). Plates are incubated at room temperature on the lab bench and used to inoculate overnight cultures which will seed experimental cultures, both in liquid medium and on agar plates.

Dr. Kreuzer-Martin stirs 48 h B. subtilis spores grown on a TSA plate with an alcohol-flamed hockey stick. The process begins with the addition of ~2 ml water to the plate surface. Once loosened, spores can be transferred to a centrifuge tube and spun to pellet.

The “loosen and transfer” process is repeated 3 times to collect as many spores as possible from the agar plates. Once the spores from one plate are combined into a single centrifuge tube, they are spun and resuspended in water to begin the washing process.

A close-up view of the transfer of loosened spores to waiting centrifuge tubes.

Here, Lesley loads spore samples in 30-ml round bottom centrifuge tubes into the centrifuge. Spores are spun and the supernatant disposed of. After resuspension in water, samples are shaken overnight in the cold room until the next wash. Washing helps to lyse and remove unwanted cell debris from the spores; the washing process is repeated each day for a minimum of 7 cycles.

A major component in surveying the isotopic variation found in bacteriological media is weighing samples for stable isotope ratio analysis. Each sample is weighed in duplicate for both O/H and C/N analyses, resulting in 4 separate weighed aliquots of each medium sample to be analyzed. Analysis weight for O/H is 200µg ± 10%, while that for C/N is 2.000mg ± 10%.