Denitrifier_Lab2_Response.txt

The following is the real situation in our lab starting from 3 years ago...

>1) How often to you visit your freezer stock?
>2) How often do you replenish your freezer with new stock?
>3) How many plates do you make before inoculation?
>4) What conditions do you grow your plates in (e.g. dark vs light, temperature).
>5) When you grab colonies from the plate, do you grab single colonies or a smear?
>6) Do you use the starter culture as in Sigman 2001?

Honestly speaking, we don't have any facility to keep denitrifier correctly. I mean, we don't have any clean benches
to innoculate denitrifer in the microbe-free environment and we don't have any good deep-freezer
to stock the denitrifer strain. So when I moved to (ANONYMOUS UNIVERSITY),
I left my denitrifer stock in (ANONYMOUS UNIVERSITY), and I started to innoculate denitrifier liquid-to-liquid
without checking the contamination with plate methods. Moreover we have not replenish the denitrifer from the
frozen stock since 2007.

I had planed to visit our freezer stock in (ANONYMOUS UNIVERSITY) when we found the accumulation of nitrite in our growth media,
but in these 3 years, we only have one occation (one bottle from 14 bottles in this case) with the NO2 accumulated.

>7) Does your growth media recipe differ from Sigman 2001 and if so, how?

We usually make 1500ml growth media with

* 45g TSB
* 7.34g KH2PO4
* 1.70g KNO3
* 0.0812g NH4Cl2

A while ago, we tried to reduce NO3 to reduce N2O blank, but we came back to this original recipe.
To reduce the N2O blank in the media, we just wash the denitrifier with new media (without nitrate).

>8) What bottle type / size do you use to grow P. aureofaciens?

We put 100ml of growth media into 100ml vial
Every week, we prepare 14 vials from 1500ml media.

We keep the ratio -- 100ml liquid in 130ml vial. We don't know how low the DO is when the growth media is 6-10days old.
But as far as we experience, this ratio (headspace vs liquid volume) works well for 3 years.

>9) How many of the bottles in #8 do you use for a single harvest?

We usually prepare 14 vials (1400ml in total). Normally we use 10-12 vials for 100samples.
 From one vial, we split the media into 2 centrifuge tube (40ml), and we use the 20ml left for NO2 concentration measurements.

>10) What conditions are the bottle(s) in when the bacteria are growing (e.g. dark vs light, temperature)?

We put the vials in the incubator (dark, 25C). My collegue told me tha incubation with low temperature can induce the
accumulation of NO2, but we are not sure, actually...

>11) What are the autoclave settings for the growth media (temperature, duration, etc)?

1hr, 121C. The media should be in "nice dark brown". The autoclaved media should be used (inoculated) asap. When we used
relatively old media (1 day after autoclaving), the growth was bad. So I usually autoclave the media in the morning, and inoculate
the media in the afternoon in the same single day.

>12) Do you use an orbital shaker or a reciprocal shaker or manual shaking?

I feel that shaking is not good -- growth was not good when we shaked the vials, so we just let the vials sit for 6-10 days.
Actually we don't shake the vials until just before the centrifuging...

>13) Can you see qualitative differences in pellets?

Among our 14 vials in each batch, the color of pellets are always pink, and the amount of pellets does not differ so much,,,

>14) How well does your method work (e.g. 50% success, 100% success, etc)?

So far almost 100% success. Even when I taught undergrad students who have never experienced microbial stuff and isotope anlaysis,
the regression curve with USGS32, 34, 35 and IAEA was good.

But, just today, our post-doc got the bad data -- d15N of USGS 32 was 150 permill. Normally d15N of USGS 32 has been measured as
165-179 permill, so something bad happens...Woops,,,,