Enabling scientific discovery through stable isotope analysis
Bacterial Denitrifier Harvesting Strategy Survey
Below is a table of various strategies used by anonymous laboratories to harvest Pseudomonas chlororaphys or Pseudomonas aureofaciens bacterial colonies for the purpose of denitrification of nitrate for δ15N and/or δ18O determination. See the original inquiry below the table. To see each laboratories response (scrubbed of any identifying information), click on the Lab Number column heading.

Lab 1Lab 2Lab 3Lab 4 Lab 5Lab 6Lab 7
1. Freezer Stock Visit-n/a-weeklyOnce per 2-6 monthsneverregularly
2. Create Freezer Stock-n/a-as needed, use stock tube 3x then discardnot yetneverregularly
3. Plates before Inoculation-n/a-2 - 31n/a3
4. Plate growth conditions-n/aambient temperatureAmbient temperature, darkAmbient temperature and lightn/a21 *C, dark
5. Colony from Plates-n/asinglesingle to multiplecouplen/asingle
6. Starter Culture-n/ayesyesyesyesyes
7. Media Recipe Different?no1.5 L, 45g TSB, 7.34g KH2PO4, 1.70g KNO3,0.0812g NH4Cl2-no antifoam until harvestnono60 g TSB, 10g K2HPO4, 2g KNO3, 2g (NH4)SO4 in 2.0 L hydro pure water, no antifoam
8. Media bottle size4/5 media, 1/5 headspace130 mL with 100 mL media500 mL, 100 mL headspace500 mL250 mL, crimp cap125 mL serum vials250 mL bottle, 202 mL media
9. Number of bottles-10-12 vials for 100 samples-1 per 25 samples8 per 40 samples64 per 40 samples
10. Media growth conditions-dark, 25 *Caway from direct sunambient temperature, light uncontrolledambient temperature and lightambient temperature and light21 *C, dark
11. Autoclave settings-1 hr, 121 *C15 min, 121 *C, 1.5 hour cycle121 *C, 30 minutesliquid cycle122 *C, 20 minutes125 *C, 20 minutes
12. Shaker typeShaker, unknown typeno shakingrecipricalorbitalorbitalmanualreciprocal
13. Pellet appearance-always pink-occasional black flecks and variable opacitylight pink, uniform sizeconsistent size and amountyes
14. Success90%near 100%-near 100%near 100%100%50-60%

The original inquiry is below:

Hi all,

I am hoping to extract as much information from you as possible regarding the exact conditions imposed on your P. aureofaciens from freezer stock to sample injection. The end result will be a table on the wiki and our web page summarizing the growth and prep conditions. The immediate need and motivation for this particular email is our survival rate has fallen off to ~25% and thus our sample throughput.

The most succinct response would be we treat our bacteria identically to Sigman 2001 or identically to Casciotti 2002. Please however, take a moment to respond to each point, recognizing the details I am asking for are not necessarily in the Sigma or Casciotti papers.

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?
7) Does your growth media recipe differ from Sigman 2001 and if so, how?
8) What bottle type / size do you use to grow P. aureofaciens?
9) How many of the bottles in #8 do you use for a single harvest?
10) What conditions are the bottle(s) in when the bacteria are growing (e.g. dark vs light, temperature)?
11) What are the autoclave settings for the growth media (temperature, duration, etc)?
12) Do you use an orbital shaker or a reciprocal shaker or manual shaking?
13) Can you see qualitative differences in pellets?
14) How well does your method work (e.g. 50% success, 100% success, etc)?

If you feel I have missed some detail that you think is particularly important, please add those items. If I receive enough of a response, I will create a table and keep labs anonymous. Reply to me directly or to the list as you see fit. Thank you for your time.

andy