. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 130 D. A. KREEGER AND C J. LANGDON The remainder of each tissue homogenate ( ml) was treated with 4 ml of 5% trichloroacetic acid (TCA), vor- texed for 30 s (Vortex-Genie, Model 12-812), heated at 90°C for 30 min, cooled in an ice bath for 30 min, and centrifuged at 1500 X g for 20 min. The supernatant was withdrawn and 1 ml used for spectrophotometric deter- mination of carbohydrate using the procedure described by Dubois et al. (1956), standardized with oyster glycogen (Sigma, Type II, G-8751) that had been subjected to
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 130 D. A. KREEGER AND C J. LANGDON The remainder of each tissue homogenate ( ml) was treated with 4 ml of 5% trichloroacetic acid (TCA), vor- texed for 30 s (Vortex-Genie, Model 12-812), heated at 90°C for 30 min, cooled in an ice bath for 30 min, and centrifuged at 1500 X g for 20 min. The supernatant was withdrawn and 1 ml used for spectrophotometric deter- mination of carbohydrate using the procedure described by Dubois et al. (1956), standardized with oyster glycogen (Sigma, Type II, G-8751) that had been subjected to the same extraction process as the tissue samples. The pellet was further treated with 3 ml M NaOH, vortexed for 10 s, heated at 90°C for 30 min, and allowed to stand overnight at room temperature. Samples were then cen- trifuged (1500 X g, 10 min), and the protein concentration of the supernatant was determined spectrophotometrically using a test kit based on the procedure of Lowry el al. (1951) (Pierce, BCA 23225). The protein content of the initial tissue homogenate was calculated from standard absorbances of purified mussel protein (for procedure, see Kreeger, 1992) that had been treated identically to the samples. Average protein, lipid, and carbohydrate contents in mussel tissues from each beaker were calculated from arcsine square root transformed data and compared sta- tistically among treatments using ANOVA and Tukey's HSD multiple range tests (Sokal and Rohlf, 1969). Results Characteristics ofmicroalgae The alga /. galbana (clone T-ISO) was successfully cul- tured in low-nitrogen (40% of the nitrogen off/2 medium, 30 mg NaNO31~') and high-nitrogen (200% of the nitro- gen off/2 medium, 150 mg NaNO31~') enriched seawater. Further reductions in nitrogen concentration below 30 mg NaNO3 r' caused algal cultures to collapse after 6 days of growth. Temporal changes in cell concentrations of low-nitrogen and high-nitrogen cultures are shown in Figure 3a. Both types of
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