PROTOCOL FOR OBTAINING AXENIC PROCHLOROCOCCUS VIA DILUTION-TOEXTINCTION USING HELPERS
J. Jeffrey Morris, May 2008
I. PRE-TREATMENT OF MEDIA WITH HELPERS (Day 1)
a. Grow up an overnight culture of Alteromonas sp. EZ55 in YTSS medium (per L:
5 g tryptone, 2.5 g yeast extract, 15 g Sigma sea salts) at ~24° C with rotation.
b. Harvest 1 mL EZ55 by centrifugation at 8000 rpm for 2 min in a microcentrifuge.
Remove supernatant by pipetting.
c. Resuspend pellet in 1 mL sterile seawater. Spin down again and remove
supernatant. Repeat this step one more time, then resuspend one last time in 1 mL
sterile seawater.
d. Place 10 L of washed EZ55 in 990 L of sterile seawater.
e. Inoculate 250 L EZ55 cells into 24.75 mL autoclaved Pro99 medium (final
concentration ~106 cells/mL).
f. Prepare another overnight EZ55 culture in YTSS.
g. Incubate this medium overnight in the dark at room temperature. During this
time, the EZ55 will deplete any hydrogen peroxide (HOOH) in the medium.
II. DILUTION-TO-EXTINCTION (Day 2)
a. You will need one 96-well tissue culture plate per Prochlorococcus strain to be
purified. Use a Falcon Microtest U-bottom plate (BD, #35-3077) or equivalent.
b. In columns 3-12 of each 96 well plate, pipet 180 L of fresh helper-treated Pro99
using a multichannel pipettor.
c. Determine concentration of Prochlorococcus culture by flow cytometry. Dilute
to approximately 5x104 cells/mL in sterile Pro99 medium.
d. Pipet 200 L of diluted Prochlorococcus culture into each well of columns 1 and
2.
e. Using a multichannel pipettor, transfer 20 L of each well in columns 1 and 2 into
corresponding wells in columns 3 and 4. Repeat this procedure for remaining
pairs of columns. The result should be 16 replicates each of 6 sequential
dilutions, ranging from 10000 cells/well to 0.1 cell/well.
f. Place cover on 96 well plate and insert into a Ziploc bag. If desired, plate may be
shielded with a piece of theater gel to achieve low-light conditions.
g. Incubate plate in appropriate incubator. 12/12 light/dark conditions at 24° C with
~40-50 E PAR work for most Prochlorococcus strains.
III. PURITY SCREEN
a. Place 200 L of YTSS containing 100 g/mL streptomycin into each well of a 96
well plate. Prepare one test plate for each plate of Prochlorococcus prepared.
These test plates may be stored in Ziploc bags at 4° C until needed. Make sure
test plates are labeled in such a way as to connect them to their sister
Prochlorococcus plate.
b. Check Prochlorococcus plates once per week. Growth is easily discerned by the
formation of green pellets at the bottoms of wells. When growth is observed in a
column, use a multichannel pipettor to transfer 20 L from every well of that
column into a corresponding test plate well.
c. Incubate the test plate in a Ziploc bag at room temperature in the dark. After
three days, any wells that have not become turbid will be considered to represent
“tentatively clean” cultures in the parent plate; i.e., they contain no streptomycinresistant heterotrophs.
IV. RECOVERY OF AXENIC PROCHLOROCOCCUS
a. For each “tentatively clean” well, prepare 5 mL of helper-treated Pro99 (see step
I) in sterile 12 mL culture tubes. Incubate overnight in the dark to eliminate
HOOH.
b. Aspirate media from “tentatively clean” Pro99 wells into these tubes. Incubate at
same light/temp conditions as parent plate.
c. Once these outgrowth cultures are clearly green (about 108 cells/mL), transfer 2
mL into 2 replicate tubes containing 2 mL each of sterile, catalase-treated Pro99.
i. Prepare 10 mL of milli-Q water buffered with 1 mM phosphate buffer, pH
7.0. Warm to 37° C.
ii. Dissolve 10 mg catalase crystals into this solution. Sterile filter using a
Millex GV PVDF syringe filter.
iii. Add sterile catalase to autoclaved Pro99 at a final concentration of 1
U/mL. Incubate at room temperature for 2-3 hours before inoculating with
Prochlorococcus.
d. Treat these cultures with a final concentration of 100 g/mL streptomycin.
e. Add 4 mL fresh catalase-treated Pro99 to original outgrowth tubes. Mark to
indicate that they have been transferred into streptomycin-treated media and
return to incubator “just in case.”
f. Incubate streptomycin-treated tubes at same light/temp conditions as parent
outgrowth tubes. After 3 or more days since antibiotic treatment, transfer 1 mL
into 3 mL purity broth (1/10 ProAC and PLAG, see Morris et al 2008) to
determine presence of remaining viable heterotrophs. This method should afford
you at least a few tubes that are both “green” and “clean.”
V. CARE AND FEEDING OF YOUNG AXENIC STRAINS
a. Initially, you will have 4 mL of the axenic strain. Odds of survival are relatively
low at this stage. Once these tubes turn noticeably green, add fresh Pro99 to the
tube 1 mL at a time, as often as once a day (provided vivid green color remains).
b. When 8 mL accumulates, transfer 4 mL into a fresh tube. Continue topping off
with 1 mL/day fresh media .
c. Once at least 3 such tubes exist, transfer the entire contents of one to a 25mm
acid-washed glass tube. Continue topping off 1 mL/day until approximately 25
mL volume is achieved.
d. At this stage, 2:1 transfers can be made every week or so (16 mL of culture into 9
mL of fresh media). Make sure to keep the old tubes and “top them off” as
before.
e. Distribute each new transfer to other lab members until everyone has a copy.