Denitrifier_Lab5_Response.txt

1.  Once every few months.  Right now it's been about 6 months and the bacteria
are still going strong.  We are about to have some extended down time on our
machine, so I will probably start a new culture when we get up and running
again.
2.  We use such a small amount to make the cultures (and do it so infrequently)
that we have not had to replenish the freezer stock yet (since we started about
2 years ago).

3.  One plate

4.  We put them in the hood and grow them at room temperature.

5.  We usually grab a couple single colonies.

6.  Yes, we follow Sigman.

7.  I think the media is exactly the same.  One thing we do that I don't know if
they do is before we inoculate the bacteria into the broth bottles, we sterilize
the caps with rubbing alcohol and a lighter.

8.  250 mL clear glass bottle with 20 mm crimp cap

9.  We use 8 and mix them all together before harvesting, but I think we really
only need 6 or 7 (for a run of 40 samples).

10.  Light (and dark at night), on a shaker table, room temperature

11. I can't find my autoclave manual right now, but it's a liquids setting that
was pre-programmed, and it gets pretty hot (the bottles are hot to the touch
when it opens).  It runs for about 20 minutes, but it takes about 30 minutes to
get to the right temperature and pressure and then it takes about an hour to
cool enough for the door to open.

12.  Orbital shaker

13.  No, they are all light pink and about the same size.

14.  We had a lot of problems getting this method started up.  One of the best
pieces of advice is to test the bacteria with nitrite test strips prior to
harvesting, to ensure that the bacteria have "eaten" all of the nitrite in the
broth.  Once we started doing that, we have not had many problems with the
bacteria.  Once in a while you will get a sample with no peak, but I would say
that is rare and can possibly be attributed to other factors (e.g. leaky bottle,
auto-sampler needle clog, etc.).  I don't think we've ever had a problem with
the bacteria dying off.