Journal of Experimental Marine Biology and Ecology 251 2000 239–263
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A new method to estimate respiration rate of biological material based on the reduction of tetrazolium violet
˚ Ulf Bamstedt
Department of Fisheries and Marine Biology , University of Bergen, P.O. Box 780, N-5020 Bergen,
Norway Received 7 December 1999; received in revised form 20 February 2000; accepted 3 May 2000
Abstract
The traditional method for measuring the activity of the electron-transport system ETS gives an estimate of the potential respiration rate, since it measures the rate under saturated substrate
conditions V . Accumulated literature data indicate that this does not relate closely to the actual
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respiration rate. The new method described here is based on reduction of tetrazolium violet in a homogenate with a natural level of substrates. The analytical protocol ensures that the spectro-
photometric reading after 1 h incubation at 408C reflects the ambient amount of substrates available. This method is superior to the traditional ETS assay in giving a closer correlation with
ambient respiration rate. A number of methodological tests have been performed and recom- mendations are given to optimize the measurements. Macrozooplankton species from the coastal
waters of Sweden, representing different taxonomic and trophic groups, showed a range in respiration ETS ratio from 1.1 to 2.9, and both inter- and intra-specific variability in this ratio
were reduced by 50–70 compared with the traditional ETS assay. Results from other environments and for particulate organic matter indicate a similar general improvement. The new
analytical assay is simple, cheap and well suited for fieldwork.
2000 Elsevier Science B.V. All
rights reserved.
Keywords : Respiration; Tetrazolium violet; Enzymatic reduction
1. Introduction
Biologists have long been using the in vitro enzymatic reduction of INT tetrazolium violet, 2- p-iodophenyl-3- p-nitrophenyl-5-phenyl tetrazolium chloride to estimate
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E-mail address : ulf.baamstedtifm.uib.no U. Bamstedt.
0022-0981 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 2 2 - 0 9 8 1 0 0 0 0 2 1 7 - 3
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240 U
. Bamstedt J. Exp. Mar. Biol. Ecol. 251 2000 239 –263
respiration rates of aquatic organisms in diverse environments e.g., Packard, 1971;
˚ Kenner and Ahmed, 1975; King and Packard, 1975; Bamstedt, 1979, 1980; Martinez,
1991, 1992, 1997; Martinez and Estrada, 1992; Hernandez-Leon and Gomez, 1996; Ikeda, 1996; Madon et al., 1998. The modified method Owens and King, 1975 gives
an estimate of the potential respiration rate, because the analytical protocol includes saturation conditions in substrate level NADH, NADPH which makes the reactions
work at the maximum rate V
. This means that the amount of available enzymes will
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be the rate-limiting step in the electron transport and the result will then be a reflection of the enzyme level in the sample. However, it is unlikely that this is the major
regulating factor in response to short-term changes in energy expenditure. A general conversion factor from ETS activity to respiration rate of 0.5 was suggested by Packard
et al. 1974 based on empirical results. The rather weak theoretical basis for this constant is that organisms would respire at around 50 of their capacity in normal
situations Packard, 1985. However, in vivo ETS activity is more likely controlled through typical enzyme kinetics, according to a Michaelis–Menten function, where the
rate is regulated by the present substrate level. This, in turn, implies that the ratio between respiration rate and ETS activity R ETS will vary with the physiological
activity of the organism. The rather wide R ETS ratios reported in the literature support
˚ this explanation see, e.g., Bamstedt, 1979; Hernandez-Leon and Gomez, 1996, for
zooplankton, although other sources of variability are certainly possible. By excluding the addition of substrates used in the original method, the measured reduction of INT
will be a result of the naturally occurring amounts of enzymes and substrates in the homogenate. Without the intact cell system that can regenerate the substrate there will be
an exponential decrease of the substrate level, causing a gradual decrease in the rate of reduction of INT as described by a Michaelis–Menten function. Theoretically, the initial
rate of reduction should give the actual ETS activity but, in practice, this is very difficult to measure, since substrate is being used up immediately when homogenisation starts,
and a considerable time is needed before a spectrophotometric reading can be taken. The alternative is to continue the reaction until total consumption of the substrates has taken
place. The spectrophotometric reading would then give a result that should be directly related to the total amount of substrates available during the incubation. Since the total
amount of substrates available will be the controlling factor for the initial activity of the ETS as defined by the Michaelis–Menten equation, the result should be a good index
of the respiration rate. In the present paper, I report on methodological tests of such a method and compare results for different marine invertebrates with this method, the
original ETS activity method as modified by Owens and King, 1975 and direct measurements of the respiration rate in incubation experiments.
2. Material and methods
