 dates:           24 January 2002 to 21 February 2002  (20020124-20020221) 
 location:        N: -52.56100  S: -66.61150  W: -172.69267  E: -166.94733

15 August 2008: Prepared for OCB data system by Cyndy Chandler, OCB DMO (WHOI) from documentation contributed by Jacques Olivier, Helen Quinby, Robert Daniels, and Hugh Ducklow

Original Excel file contributed: 8 November, 2002

and downloaded from MBARI by OCB DMO:
copy of original Excel file

Contact: Jacques Oliver (Email: jloliver@vims.edu)

R/V Melville Bacterial Measurements Methodology

Bacterial Abundance Flow cytometry (FCM)

1. 9.5 ml sample from CTD or TM rosette, fixed to final concentration of 1% formaldehyde

2. Sample frozen at -80 C

3. On land, triplicate samples were analyzed using a Beckman-Coulter Epics Altra equipped with an Enterprise II laser at 488 nm using 190mV (intra-sample variation).

4. About 100 ml of sample were measured following Troussellier et al. (1999) with addition of 2.5 mM SYTO 13 and a 10 minute incubation.

5. This analysis was done on triplicate 1.0 ml samples to which 1.0 mm beads (Molecular Probes, Fluo Spheres, F-8888) were also added at a 10E6/ml concentration.

The beads determined flow rate and uniformity in fluorescence signal. Discrimination was done on a positive green fluorescence and the plot was green fluorescence (devoid of any red fluorescence) against side scatter. Analytical variation was 3%.

Reference: Troussellier, M., C. Courties, P. Lebaron, and P.Servais. 1999. FEMS Microbiology Ecology 29: 319-330.

Bacterial Abundance Acridine orange direct counts (AODC)

  1. 10-15 ml sample fixed to final concentration of 1% formaldehyde               		
  2. Sample filtered onto 0.2 micron blackened polycarbonate filter               		
  3. Filter stained with 45% (w/v) acridine orange                		
  4. Filter mounted onto glass slide with immersion oil and coverslip               		
  5. Bacteria enumerated via epifluorescence microscopy               		
  6. Intra-sample variation reported

Reference: Kirchman, D., Sigda, J., Kapuscinski, R., and Mitchell, R. 1982. Appl. Envrion. Microbiol. 44:376-382

Bacterial Production Thymidine (TdR) and Leucine (Leu) Incorporation

   1. 1.6 ml sample spiked with tritiated thymidine or tritiated leucine               		
   2. Samples run in triplicate with one negative control containing 6% trichloroacetic acid (TCA)               		
   3. Samples incubated at in situ temperatures in the dark for 8-20 hours (depending on water temp)               		
   4. Incubation stopped by adding TCA (6% final conc.) to each sample               		
   5. Nucleic acid extracted with TCA and ethanol               		
   6. 1.6 ml Ultima Gold scintillation cocktail added               		
   7. 3 minute counts on each sample on scintillation counter

Reference: Smith, D.C. and Azam, F. 1992. Mar. Microb. Food Webs. 6:107-114.

bacteria.csv