Bacterioplankton – 03 Results – Isolation, culturability, and morphological data

Representative Freshwater Bacterioplankton Isolated from Crater Lake, Oregon

Kathleen A. Page, Stephanie A. Connon, and Stephen J. Giovannoni

Results

Isolation, culturability, and morphological data. Crater Lake is known for its extreme clarity and typically has deep chlorophyll maxima (25). Maximum chlorophyll concentration was observed at 130 m on the day of sampling. Bacterioplankton concentrations at 15, 130, 300, and 500 m were determined to be 2.4 105, 3.5 105, 1.3 105, and 0.7 105 cells/ml, respectively. Lake temperature below 100 m is approximately 3.5°C year-round and from 4 to 17°C at the surface, and dissolved oxygen is present throughout the water column (25, 28, 44). In an attempt to mimic natural conditions, cultures were initiated aerobically at 4, 10, and 15°C in the dark. Key features of the high-throughput culturing (HTC) method used in this study include culture media consisting of sterilized natural water amended with very low levels of organic and mineral nutrients and extinction culturing in liquid media to obtain pure cultures (7, 37). Isolates cultured in mixed carbon or yeast extract-acetate media were obtained from lake depths of 15, 130, 300, and 500 m, and details are summarized in Table 1. One hundred fifty-three of 672 extinction culture wells were positive for bacterial growth. Culturability ranged from 0.1 to 2.6%, with an overall average of 0.8%. This low rate of culturability may reflect an intrinsic inability of highly diverse populations to grow in a single medium, a high frequency of dormant or noncultivable cells, nutrient limitations in the media used, or inhibitory culture conditions. Organisms collected from 15 and 130 m had an average culturability of 1.8%, whereas organisms from 300 and 500 m had an average culturability of 0.6%.

No. of isolates at specific depths (m)
Isolate HTCC
no(s).
Division 15 130 300 500 Growth in
10% R2A
Morphology and dimensions
500-502 Alphaproteobacteria 2 1 Rod, 0.5 1.0 m
503-513 Alphaproteobacteria 8 3 Rod, 0.5 0.8 m
515-517 Alphaproteobacteria 3 Rod, 0.5 1.5 m
518-524 Betaproteobacteria 7 + Rod, 0.5 2.5 m
525-527 Betaproteobacteria 3 Vibrio, 0.6 3.1 m
528-539 Betaproteobacteria 1 5 5 1 Rod, 0.7 3.5 m
540 Betaproteobacteria 1 Rod, 0.5 5.0 m
541-542 Betaproteobacteria 1 1 Rod, 0.7 8.1 m
543 Betaproteobacteria 1 Coccus, 0.5- m diameter
544-549 Gammaproteobacteria 5 1 + Rod, 0.7 1.0 m
550 Gammaproteobacteria 1 + Rod, 0.5 1.1 m
551 Gammaproteobacteria 1 Coccobacillus, 0.5 0.7 m
552 Bacteroidetes 1 + Rod, 0.7 4.7 m
553 Bacteroidetes 1 + Rod, 0.7 4.4 m
554 Bacteroidetes 1 Filament, 0.5 8.5 m
555 Actinobacteria 1 +

Rod, 0.5 1.0 m

TABLE 1. Classification and isolation and culture characteristics of 55 Crater Lake isolates in 16 groups