Diagram  

+ click to enlarge

Conceptual Diagram of Microbial Growth

Phase 

Protocol for Bacterial Heterotrophic production

• 10 place filter manifold

• Towers (chilled overnight @ 4C)

• 5% (w/v) Trichloroacetic acid (TCA) (chilled overnight @ 4C) Usually 1L is enough for a single time series set

• 32 15ml centrifuge tubes

• Pump

• Scintillation vials and fluid (UltimaGold)

• Lab supplies

^Top

• Label in duplicate, 4 15ml centrifuge tubes for each depth (3 samples and 1 control). One set for Thymidine and another for Leucine.

• Rinse each tube 3x with ~5ml of sample seawater

• Pour 10ml of sample into rinsed centrifuge tube

• Kill control samples by adding 0.1ml of filtered formalin. Let stand 10’.

• Pipette 5nM of tritiated Thymine or 5nM of tritiated Leucine into sample. Typically vol. TdR<vol Leu. Dispose of tips into labeled plastic whirlpak bags

• Incubate samples at seawater temperature for 1 hour.

• During incubation, label scintillation vials.

• 10’ before incubation time ends set-up Filtration manifold with 0.45um HA Millipore filters. Place chilled towers.

• Following incubation pour samples into respective wells and filter through (<0.3 atm). Make sure no leaks!!

• Turn off pump and close wells.

• Add ~2ml of 5% TCA to each well. Let stand for 2’

• After 2’, open wells and filter through TCA

• Rinse towers 3x with 5% TCA

• Remove towers (pump is still on!)

• Rinse filter 3x with 5% TCA, enough to cover filters completely

• Turn off pump

• Remove filters and place in labelled scintillation vials

• Add 5ml of UltimaGold scintillation fluid

• Tightly cap scint vial and shake HARD; make sure the filter ends up in the liquid

• Let sit for at least 3 hours before final counts, preferably overnight

• Perform wipetest of surrounding area (see map)

• Read samples in scintillation counter. Currently using program #1 (1’ read)

^Top

• Rinse towers with diH2O

• Rinse top of Filtration manifold with diH2O

• Pour radioactive filtrate out of Filtration manifold into waste container carefully (use a funnel!)

• Throw dry waste into radioactive waste barrel (not the regular trash!)

• Record isotope usage online

^Top

Related Links