Denitrifier_Lab7_Response.txt

*1) How often to you visit your freezer stock? *

*
*We normally start each cultivation cycle with a freezer stock of 
bacteria. We open a freezer stock sample, take 5 ?l out with a sterile 
pipette and transfer it on a plate, mark the tube as used, freeze it 
again and use it again in the next cycle until it is finshed.

*2) How often do you replenish your freezer with new stock?*

* *

In the beginning we replenished our freezer with a new stock each time 
we started a new cycle. Meanwhile (they became far too many) we do that 
every fourth or fifth time.


*3) How many plates do you make before inoculation? *

*
*We make new plates every 14 days and store them in a fridge. They last 
for about two 2 cycles.

We use three plates (#1, #2, #3) and each 2 plates (if one is no 
good...) before inoculation.

*4) What conditions do you grow your plates in (e.g. dark vs light, 
temperature)? *

We cultivate our plates in the dark in an inoculation cupboard at a 
constant temperature of 21?.


*5) When you grab colonies from the plate, do you grab single colonies or a
smear?*

We grab a single colony which means a single well defined dot.

*6) Do you use the starter culture as in Sigman 2001? *

Yes. After reviving bacteria cultures on #1 and transferring to #2 and 
#3 we transfer a single colony to a tube with 5ml nutrient broth (1,6 g 
TSB in 200 ml hydro pure water). It grows overnight on the shaker in the 
inoculation cupboard.


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

Our medium is compound of 60 g TSB, 10 g K_2 HPO_4 , 2 g KNO_3 and 2 g 
(NH_4 )SO_4 in 2,0 l hydro pure water. We do not ad antifoaming agent.

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

We started to incubate /P. aureofaciens/ in 200 ml (nominal volume, true 
volume 250 ml) bottles with about 166 ml of medium. Bacteria seemed to 
grow well and never smelled bad. However, isotope data showed an 
unsatisfied high standard deviation and it seemed that bacteria did not 
work properly so that measured values were way too high for the IAEA N3 
and USGS 34 standards. Now we increased the medium volume up to 202ml 
(but we are still using the same bottles) to raise the medium to air 
supernatant ratio in the bottles. Bacteria "work" perfectly now. 
Standard deviation of international reference material is within 0.20/00 
for ?15N and 0.5 for ?18O. However, now we do have the problem, that 
almost every other day the bacteria are not working well and we obtain 
way to high peaks in the MS. It seems that the bacteria do not reduce 
the nitrate from the medium but it is hard to tell from the smell which 
flask is good and which one is not. We did several times the nitrite 
test to check whether there is still nitrite in the medium bottles, but 
still if it is negative we might get the "high peak phenomenon"....

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

We use four bottles a day for 40 samples.

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

Bottles are in the same incubator cupboard as the bacteria plates: in 
the dark and with constant temperature of 21?C.

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

We do autoclave the medium (first and second) at 125?C for 20 min (small 
table autoclave) and the medium bottles for inoculation at 121?C for 30 
min in a big lab autoclave.

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

It's a reciprocal shaker. We increased the shaking to ~180 rpm so that 
media get really foamy.

*13) Can you see qualitative differences in pellets? *

Yes. When we had really smelly (stabbing) medium in the bottles after 
the incubation the pellets were rather small or they did not really 
become a pellet but remained sticky on the walls of the tube. However, 
we do have pellet which look perfectly but still peaks in the MS is much 
too high (see above) and isotope measurement is then nonsense....


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

50-60%

We had a lot of problems in the beginning and we did not know where and 
what to change. E.g. not only that our standard deviation was too high, 
but we did not really "hit" the accepted value for the international 
standards such as IAEA N3 and USGS 34.

However, we changed our N_2 O bottle since we did not know if it was 
rather empty. That helped a lot to measure accepted values and to 
stabilize the ratios for the standards. The most important thing we 
changed was the media volume from 166 to 202 ml. And we increased the 
shaking form 160 to 180 rpm.