Determination of Microbial Production
179
thymidine was more than three times less than the nominal one written on its
batch label. Now how could I believe the nominal specific radioactivity of this
batch? The specific radioactivity written on the batch is, however, the main
parameter used for the calculation of TDR uptake and microbial production.
The researcher himself has no possibility of controlling it because its specific
radioactivity is too high: the whole batch contains some O.5llmol of thymidine.
In this situation I decided to calibrate the thymidine uptake rate, expressed as
cpm per lOOml of sampleh- 1 after the CO2 dark uptake method. When measuring the microbial production in the coral reef waters simultaneously by
both methods there was good coincidence between them in most of the stations; in some of them, however curiously high values were obtained by the
thymidine method, inexplicable from the point of view of the bacterioplankton biomass background. So, finally, I abandoned the thymidine method and
returned to the safer, in my opinion, 14C02 dark uptake one. The same story
repeated itself when I made another attempt to apply the thymidine method
to measure microbial production in Antarctic waters.
The thymidine methodology was severely criticized even by Dr. D. Karl,
who was among its main users and interpreters (Karl 1986).
In any case, we need to measure the microbial production now without
expecting the appearance of an "ideal" method. Therefore it is necessary
to realize the weak and strong sides of the existing methods in order to use
them in an aware and informed manner and not just "grin and bear it". The
latter often happens, when people use a methodology blindly and pay no attention to its known drawbacks. The methods technically convenient but difficult
to interpret have to be calibrated after another, maybe older but safer,
methodology.
At present we have a selection of methods for the assessment of microbial production, but maybe only two of them have a solid background and
minimal drawbacks, so they can be used as reference methods for calibration
of other methods technically simpler and more sensitive but with drawbacks
which make the results difficult to interpret and less versatile. Ivanov's method
(1955), based on direct microscopic analysis of microbial growth rate in water
samples, and the radioisotopic 14C-dark uptake method by Romanenko (1963,
1964) can be recognized as such reference methods. Both of them since have
been significantly modified during long practice of their use. The application
of epifluorescence microscopy for direct counting of bacteria made the direct
microscopic method more precise and reliable. For the improvement of 14C
dark uptake method, the appearance of Nucleopore filters was very important, to separate bacterioplankton from phytoplankton. Both these methods
also have their drawbacks; these drawbacks are understandable and open to
correction and control, which makes them reliable in good hands, because their
use needs a definite art and careful approach.
The thymidine (TDR) method proposed by Fuhrman and Azam (1982) is
technically very simple, rapid, and sensitive. Its use practically eliminates problems of bottle effects and grazing losses. These features, as well as a high se!ec-
179
thymidine was more than three times less than the nominal one written on its
batch label. Now how could I believe the nominal specific radioactivity of this
batch? The specific radioactivity written on the batch is, however, the main
parameter used for the calculation of TDR uptake and microbial production.
The researcher himself has no possibility of controlling it because its specific
radioactivity is too high: the whole batch contains some O.5llmol of thymidine.
In this situation I decided to calibrate the thymidine uptake rate, expressed as
cpm per lOOml of sampleh- 1 after the CO2 dark uptake method. When measuring the microbial production in the coral reef waters simultaneously by
both methods there was good coincidence between them in most of the stations; in some of them, however curiously high values were obtained by the
thymidine method, inexplicable from the point of view of the bacterioplankton biomass background. So, finally, I abandoned the thymidine method and
returned to the safer, in my opinion, 14C02 dark uptake one. The same story
repeated itself when I made another attempt to apply the thymidine method
to measure microbial production in Antarctic waters.
The thymidine methodology was severely criticized even by Dr. D. Karl,
who was among its main users and interpreters (Karl 1986).
In any case, we need to measure the microbial production now without
expecting the appearance of an "ideal" method. Therefore it is necessary
to realize the weak and strong sides of the existing methods in order to use
them in an aware and informed manner and not just "grin and bear it". The
latter often happens, when people use a methodology blindly and pay no attention to its known drawbacks. The methods technically convenient but difficult
to interpret have to be calibrated after another, maybe older but safer,
methodology.
At present we have a selection of methods for the assessment of microbial production, but maybe only two of them have a solid background and
minimal drawbacks, so they can be used as reference methods for calibration
of other methods technically simpler and more sensitive but with drawbacks
which make the results difficult to interpret and less versatile. Ivanov's method
(1955), based on direct microscopic analysis of microbial growth rate in water
samples, and the radioisotopic 14C-dark uptake method by Romanenko (1963,
1964) can be recognized as such reference methods. Both of them since have
been significantly modified during long practice of their use. The application
of epifluorescence microscopy for direct counting of bacteria made the direct
microscopic method more precise and reliable. For the improvement of 14C
dark uptake method, the appearance of Nucleopore filters was very important, to separate bacterioplankton from phytoplankton. Both these methods
also have their drawbacks; these drawbacks are understandable and open to
correction and control, which makes them reliable in good hands, because their
use needs a definite art and careful approach.
The thymidine (TDR) method proposed by Fuhrman and Azam (1982) is
technically very simple, rapid, and sensitive. Its use practically eliminates problems of bottle effects and grazing losses. These features, as well as a high se!ec-