These were incubated anaerobically at 85°C and 1 atm, 110°C and slight gas (H2CO2)
pressure, or in glass syringes in pressure vessels at 98°C and 265 atm hydrostatic pressure.
Additional sources of inocula included external scrapings and gut contents of a polychaete worm (Alvinella sp.) found buried within its organic tube in the smoker rock 1-2
cm from an exit conduit, macerated bits of the worm tube peeled away from the rock after
breaking it open, and scrapings from the underlying rock surfaces after both the worm
and its tube had been removed. Visual differences (changes in indicator dyes, yellow
sulfur crystals, or turbidity) and differences in gas production (increased gas pressure, the
smell of H2S) between samples and sterile controls indicated potential microbial activity
in 17 of 91 inoculated samples. Of these, the most frequent source of inoculum was the
black-smoker effluent itself or scrapings from the smoker rock. A few positive reactions
were detected for Alvinella tube samples and external scrapings, but not for gut samples.
The most common medium showing positive indications of activity was a sulfur-based
medium, formulated according to Baross, Deming, and Becker (1984), or a medium
prepared shipboard using.2µm-filtered black smoker effluent supplemented with 0.03 %
(NH 4 ) 2 SO 4 , 0.01 % NaHCO 4 , and 0.5 % flowers of sulfur. Activity was apparent under
the most favorable incubation conditions (anaerobic, 110°C, slight gas H 2 / CO 2 pressure)
18-72 h after inoculation.
After the cruise, enrichments were transported back to the lab in a Dewar flask containing
hot sand and boiling water, which measured 60°C upon arrival. Transfers of the presumptive cultures into fresh media resulted in positive activity for 6 of the 17 samples. Of these
6, only 2 transferred a second time. Further attempts to revive these remaining cultures
have not been successful.
Field samples fixed in 2 % glutaraldehyde immediately after collection were also examined by acridine orange staining and epifluorescence microscopy. The hot water samples,
which required > 10 -3 dilution to minimize interfering orange fluorescence from angular
particles, contained about 10 5 yellow or green- fluorescing bacteria-like structures ml .
Surrounding ambient and low temperature (<40°C) vent waters, which did not require
dilution and could be counted more accurately, contained 6.3 x 104 to 1.3 x 106 bacteria
ml'
1 . An incredible biomass of widely diverse bacterial morphologies, including 8-x
100-µm filaments, was observed on the internal surfaces of the tube produced by the
Alvinella worm. A similar filamentous microorganism was detected microscopically in
one of the enrichment cultures, but failed to grow upon transfer. In spite of difficulties in
obtaining cultures of bacteria from these various samples, the microscopic work indicated
the presence of significant bacterial populations in every smoker-related sample.
In August of 1984, six samples of hydrothermal fluid, ranging in temperature from 15°C
to 345°C, were collected in goldlined titanium samplers by Alvin along the Juan de Fuca
Ridge off the coast of Oregon and Washington. Fixed and unfixed portions of these
samples, which had been removed from the samplers and handled under strictly anaerobic conditions, were made available to me by Baross. Concentrations of bacteria in these
samples, determined by epifluorescence microscopy (using acridine orange and DAPI
stains), ranged from 3.8 x 10 5 to 7.1 x 10 6 ml -1 with the highest level detected in the hottest
(345°C) sample (Deming and Baross, 1986).
More success was obtained in culturing bacteria from these samples than from samples
collected at 13°N. We devised a new solid medium, based on the highly thermostable
solidifying agent GELRITE (Kelco, San Diego, CA), that contained 0.5 % NaAcetate and
0.3 % thiosulfate in addition to various sea salts and trace elements. It was inoculated in
liquid pour tubes at 95°C, solidified by slight cooling to about 80°C, and incubated in
329
pressure, or in glass syringes in pressure vessels at 98°C and 265 atm hydrostatic pressure.
Additional sources of inocula included external scrapings and gut contents of a polychaete worm (Alvinella sp.) found buried within its organic tube in the smoker rock 1-2
cm from an exit conduit, macerated bits of the worm tube peeled away from the rock after
breaking it open, and scrapings from the underlying rock surfaces after both the worm
and its tube had been removed. Visual differences (changes in indicator dyes, yellow
sulfur crystals, or turbidity) and differences in gas production (increased gas pressure, the
smell of H2S) between samples and sterile controls indicated potential microbial activity
in 17 of 91 inoculated samples. Of these, the most frequent source of inoculum was the
black-smoker effluent itself or scrapings from the smoker rock. A few positive reactions
were detected for Alvinella tube samples and external scrapings, but not for gut samples.
The most common medium showing positive indications of activity was a sulfur-based
medium, formulated according to Baross, Deming, and Becker (1984), or a medium
prepared shipboard using.2µm-filtered black smoker effluent supplemented with 0.03 %
(NH 4 ) 2 SO 4 , 0.01 % NaHCO 4 , and 0.5 % flowers of sulfur. Activity was apparent under
the most favorable incubation conditions (anaerobic, 110°C, slight gas H 2 / CO 2 pressure)
18-72 h after inoculation.
After the cruise, enrichments were transported back to the lab in a Dewar flask containing
hot sand and boiling water, which measured 60°C upon arrival. Transfers of the presumptive cultures into fresh media resulted in positive activity for 6 of the 17 samples. Of these
6, only 2 transferred a second time. Further attempts to revive these remaining cultures
have not been successful.
Field samples fixed in 2 % glutaraldehyde immediately after collection were also examined by acridine orange staining and epifluorescence microscopy. The hot water samples,
which required > 10 -3 dilution to minimize interfering orange fluorescence from angular
particles, contained about 10 5 yellow or green- fluorescing bacteria-like structures ml .
Surrounding ambient and low temperature (<40°C) vent waters, which did not require
dilution and could be counted more accurately, contained 6.3 x 104 to 1.3 x 106 bacteria
ml'
1 . An incredible biomass of widely diverse bacterial morphologies, including 8-x
100-µm filaments, was observed on the internal surfaces of the tube produced by the
Alvinella worm. A similar filamentous microorganism was detected microscopically in
one of the enrichment cultures, but failed to grow upon transfer. In spite of difficulties in
obtaining cultures of bacteria from these various samples, the microscopic work indicated
the presence of significant bacterial populations in every smoker-related sample.
In August of 1984, six samples of hydrothermal fluid, ranging in temperature from 15°C
to 345°C, were collected in goldlined titanium samplers by Alvin along the Juan de Fuca
Ridge off the coast of Oregon and Washington. Fixed and unfixed portions of these
samples, which had been removed from the samplers and handled under strictly anaerobic conditions, were made available to me by Baross. Concentrations of bacteria in these
samples, determined by epifluorescence microscopy (using acridine orange and DAPI
stains), ranged from 3.8 x 10 5 to 7.1 x 10 6 ml -1 with the highest level detected in the hottest
(345°C) sample (Deming and Baross, 1986).
More success was obtained in culturing bacteria from these samples than from samples
collected at 13°N. We devised a new solid medium, based on the highly thermostable
solidifying agent GELRITE (Kelco, San Diego, CA), that contained 0.5 % NaAcetate and
0.3 % thiosulfate in addition to various sea salts and trace elements. It was inoculated in
liquid pour tubes at 95°C, solidified by slight cooling to about 80°C, and incubated in
329