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 which was posted circa 2010. To see each laboratories response (scrubbed of any identifying information), click on the Lab Number column heading.
|Lab 1||Lab 2||Lab 3||Lab 4||Lab 5||Lab 6||Lab 7|
|1. Freezer Stock Visit||-||n/a||-||weekly||Once per 2-6 months||never||regularly|
|2. Create Freezer Stock||-||n/a||-||as needed, use stock tube 3x then discard||not yet||never||regularly|
|3. Plates before Inoculation||-||n/a||-||2 - 3||1||n/a||3|
|4. Plate growth conditions||-||n/a||ambient temperature||Ambient temperature, dark||Ambient temperature and light||n/a||21 *C, dark|
|5. Colony from Plates||-||n/a||single||single to multiple||couple||n/a||single|
|6. Starter Culture||-||n/a||yes||yes||yes||yes||yes|
|7. Media Recipe Different?||no||1.5 L, 45g TSB, 7.34g KH2PO4, 1.70g KNO3,0.0812g NH4Cl2||-||no antifoam until harvest||no||no||60 g TSB, 10g K2HPO4, 2g KNO3, 2g (NH4)SO4 in 2.0 L hydro pure water, no antifoam|
|8. Media bottle size||4/5 media, 1/5 headspace||130 mL with 100 mL media||500 mL, 100 mL headspace||500 mL||250 mL, crimp cap||125 mL serum vials||250 mL bottle, 202 mL media|
|9. Number of bottles||-||10-12 vials for 100 samples||-||1 per 25 samples||8 per 40 samples||6||4 per 40 samples|
|10. Media growth conditions||-||dark, 25 *C||away from direct sun||ambient temperature, light uncontrolled||ambient temperature and light||ambient temperature and light||21 *C, dark|
|11. Autoclave settings||-||1 hr, 121 *C||15 min, 121 *C, 1.5 hour cycle||121 *C, 30 minutes||liquid cycle||122 *C, 20 minutes||125 *C, 20 minutes|
|12. Shaker type||Shaker, unknown type||no shaking||reciprical||orbital||orbital||manual||reciprocal|
|13. Pellet appearance||-||always pink||-||occasional black flecks and variable opacity||light pink, uniform size||consistent size and amount||yes|
|14. Success||90%||near 100%||-||near 100%||near 100%||100%||50-60%|
The original inquiry is below:
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.