tions can be misleading in that a succession of predominant organisms will occur as the
conditions change during the culture. These disadvantages have been discussed by
Jannasch (1967).
A continuous culture system on the other hand allows bacteria to be cultured under
controlled conditions at very low nutrient levels — more akin to those encountered in the
natural environment — and at low growth rates which can be varied independently of the
temperature. Perhaps more importantly for this type of work, it is possible using
continuous cultures to isolate defined communities or associations of organisms growing
on complex organic substrates (Cossar et al. 1981, Harder 1981, Senior et al. 1976, Slater
1978). By varying the conditions of the enrichment it is possible to isolate different
associations from the same environment, giving some indication of both the spectrum of
organisms present and their inter-relationships (Dunn et al. 1980).
Although the experimental systems used often appear complex they are simple to operate
and we have found that if mounted on a gimbal table such apparatus can be run
effectively on board ships while at sea (Brown and Warded 1983).
EXPERIMENTAL
The experiments described are enrichments of water and sediment from a marine
environment carried out in the presence of the biocide Dodigen (Hoescht Ltd) —
chemical name coco-propylene diamine guanidinium acetate — which has properties
very similar to those of a quaternary ammonium compound. Four chemostats were
employed using a carbon-limited artificial sea water medium. The four experimental
regimes were :
A No carbon source provided - as a control
B Acetate at 5mM
C Acetate at 5mM + 10 ppm Dodigen 181-1
D 100 ppm Dodigen 181-1 as sole source of carbon and energy.
The culture vessels were inoculated with a slurry of sediment and water collected from a
depth of 33 m on the continental shelf between Malin Head and the Outer Hebrides.
After inoculation, medium was metered in to give a dilution rate of 0.05 h 1 . The cultures
were continuously stirred and aerated during the course of the experiments and the
culture temperature maintained at 5° Celsius. The cultures were sampled daily, serial
dilutions prepared and inoculated onto 3 different media. These were Artificial Sea
Water Agar containing 5mM acetate as carbon source (ASWA), ASWA + 10 ppm
Dodigen and ASWA + 10 ppm Dodigen. Plates were incubated at 5°C until mature
colonies had developed after about 6-10 days. Viable counts were performed and the
results are given in Table 1.
The control experiment (A) provided useful information, it will be noted that there was
an increase in the population during the course of the enrichment although no carbon
and energy source was provided. This must be attributed to traces of carbon compounds
present in the other chemicals used to prepare the medium and gives some indication of
the background population to be expected. Note also that the presence of the biocide in
the plating medium had a depressing effect on the population, although not as great as
might have been expected. Also, although at no time exposed to the biocide during
culture, organisms tolerant to 100 ppm Dodigen were always recovered suggesting that
this was an inherent property of the heterotrophic microbial population. These results
are confirmed by the second enrichment (B Table 1) with 5mM acetate, the essential
difference here being the elevated populations due to the provision of a carbon and
energy source.
630
conditions change during the culture. These disadvantages have been discussed by
Jannasch (1967).
A continuous culture system on the other hand allows bacteria to be cultured under
controlled conditions at very low nutrient levels — more akin to those encountered in the
natural environment — and at low growth rates which can be varied independently of the
temperature. Perhaps more importantly for this type of work, it is possible using
continuous cultures to isolate defined communities or associations of organisms growing
on complex organic substrates (Cossar et al. 1981, Harder 1981, Senior et al. 1976, Slater
1978). By varying the conditions of the enrichment it is possible to isolate different
associations from the same environment, giving some indication of both the spectrum of
organisms present and their inter-relationships (Dunn et al. 1980).
Although the experimental systems used often appear complex they are simple to operate
and we have found that if mounted on a gimbal table such apparatus can be run
effectively on board ships while at sea (Brown and Warded 1983).
EXPERIMENTAL
The experiments described are enrichments of water and sediment from a marine
environment carried out in the presence of the biocide Dodigen (Hoescht Ltd) —
chemical name coco-propylene diamine guanidinium acetate — which has properties
very similar to those of a quaternary ammonium compound. Four chemostats were
employed using a carbon-limited artificial sea water medium. The four experimental
regimes were :
A No carbon source provided - as a control
B Acetate at 5mM
C Acetate at 5mM + 10 ppm Dodigen 181-1
D 100 ppm Dodigen 181-1 as sole source of carbon and energy.
The culture vessels were inoculated with a slurry of sediment and water collected from a
depth of 33 m on the continental shelf between Malin Head and the Outer Hebrides.
After inoculation, medium was metered in to give a dilution rate of 0.05 h 1 . The cultures
were continuously stirred and aerated during the course of the experiments and the
culture temperature maintained at 5° Celsius. The cultures were sampled daily, serial
dilutions prepared and inoculated onto 3 different media. These were Artificial Sea
Water Agar containing 5mM acetate as carbon source (ASWA), ASWA + 10 ppm
Dodigen and ASWA + 10 ppm Dodigen. Plates were incubated at 5°C until mature
colonies had developed after about 6-10 days. Viable counts were performed and the
results are given in Table 1.
The control experiment (A) provided useful information, it will be noted that there was
an increase in the population during the course of the enrichment although no carbon
and energy source was provided. This must be attributed to traces of carbon compounds
present in the other chemicals used to prepare the medium and gives some indication of
the background population to be expected. Note also that the presence of the biocide in
the plating medium had a depressing effect on the population, although not as great as
might have been expected. Also, although at no time exposed to the biocide during
culture, organisms tolerant to 100 ppm Dodigen were always recovered suggesting that
this was an inherent property of the heterotrophic microbial population. These results
are confirmed by the second enrichment (B Table 1) with 5mM acetate, the essential
difference here being the elevated populations due to the provision of a carbon and
energy source.
630