Denitrifier_Lab4_Response.txt

1) We revisit our freezer stock each time we begin the procedure (roughly
1x/week).

2) We replenish our freezer stock as needed. Generally, we freeze 7-12
individual vials of P. aureofaciens at once, and access each vial at most 3
times before discarding. As a result, we tend to replenish our freezer stock
every 4-7 months.

3) We make 2 to 3 plates before inoculation.

4) We grow our plates at room temperature in the dark.

5) We aim to grab single colonies, but often end up grabbing multiple
colonies from a single area.

6) Yes - We grow starter cultures of P. aureofaciens in 5 mL vials on an
orbital shaker. The time for growing our starter culture varies: between
6-30 hours.

7) We do not add antifoaming agent to our growth media. We only add antifoam
on sample prep day, after centrifuging and resuspending the bacteria.

8) We use 500 mL Pyrex bottles.

9) We use 1-500 mL bottle for every 25 vials needed. Since we prepare vials
in batches of ~50 or ~100, that amounts to either 2 or 4 bottles per
harvest.

10) Bottles are kept at room temperature, in varying conditions of
light/dark.

11) We autoclave our growth media at 121 deg C for 30 minutes (up to 50
minutes for large batches).

12) An orbital shaker.

13) Occasionally we see slight qualitative differences in pellets with
respect to size, color (presence of black 'flecks'), and opacity. If a
pellet is >50% translucent, we consider the culture unsuccessful and start
over. However, for the most part our pellets are consistent in appearance.

14) With regard to bacteria viability through harvest, our success rate is
near 100%. However, we sometimes encounter other issues, such as high method
blanks. We recently increased flushing time from 2 to 4 hours in order to
reduce our method blanks.