Determination of Microbial Production
217
by addition of the E. coli suspension-labeled CH)-thymidine into the sample
of sediment and subsequent recovery of its DNA radioactivity after the extraction procedure. Calculations of bacterial production are usually performed
in the same way as described above for water. First, the amount of CH)thymidine incorporated is calculated using the nominal specific radioactivity
of the batch, WhICh IS sometimes corrected for isotopic dilutiun according to
Moriarty's method (1990) and accounting for the recovery efficiency of DNA.
Then the number of bacterial cells produced is calculated as in experiments
with water samples.
Taking into account the numerous obscure aspects of this methodology,
like rapid CH)-TDR catabolism in sediments, a large nonspecific labeling, the
possibility of DNA labeling via the TDR catabolism and de novo synthesis
pathway, the indefinite character of specific radioactivity of CH)-TDR and of
theoretical or assumed conversion factors (the example of 2 orders of value
shifts with the bacterioplankton), it is difficult to recognize this way of using
R; values for calculation of bacterial production. The only way to obtain realistic values can be seen in its intercalibration. Because the use of the FDC
method has been disproved for bottom sediments, the dark 14COZ uptake
method in its modern version described above constitutes the only realistic
alternative. The principle of intercalibration is the same as described above for
water. The conversion factor K f between R; and bacterial production (Pb),
expressed in units of mg C cpm- I cm- 3 h- I is calculated as a result of estimation
of both values in parallel experiments. Then the R; values measured as cpm
1 cm- 3 h- I during routine experiments with CH)-TDR can be converted into
units of bacterial production: Pb = R;K f 24 g C cm- 3 day-I.
4.4.4 The Labeled P04P - Uptake Method
The possibility of using the inorganic phosphate uptake rate to estimate microbial growth was mentioned by Karl (1982) and was practically realized by Karl
and Bossard (1985), who approached the calculation of the specific growth
rate of microbial popUlations by estimating the turnover rate of the nucleotide
pool in this way. Moriarty (1990) proposed measuring microbial production in
sediments via estimation of phospholipid synthesis with the aid of labeled
phosphate 3Zp04-p' The labeled phosphate which is added into the natural
water samples is readily consumed by bacteria and incorporated into the most
important constituents of their cells such as nucleic acids, ATP, and phospholipids. I used to measure bacterioplankton production with the aid of 33p04_p
simply by estimating total uptake of P04-P by natural microbial popUlations
separated from the phytoplankton by a special filtration procedure. This
approach was based upon the assumptions that: (1) the internal pool of P04P in the cells of bacterioplankton is negligible in comparison with the external one, and (2) that the use of external P04-P in bacterioplankton is
217
by addition of the E. coli suspension-labeled CH)-thymidine into the sample
of sediment and subsequent recovery of its DNA radioactivity after the extraction procedure. Calculations of bacterial production are usually performed
in the same way as described above for water. First, the amount of CH)thymidine incorporated is calculated using the nominal specific radioactivity
of the batch, WhICh IS sometimes corrected for isotopic dilutiun according to
Moriarty's method (1990) and accounting for the recovery efficiency of DNA.
Then the number of bacterial cells produced is calculated as in experiments
with water samples.
Taking into account the numerous obscure aspects of this methodology,
like rapid CH)-TDR catabolism in sediments, a large nonspecific labeling, the
possibility of DNA labeling via the TDR catabolism and de novo synthesis
pathway, the indefinite character of specific radioactivity of CH)-TDR and of
theoretical or assumed conversion factors (the example of 2 orders of value
shifts with the bacterioplankton), it is difficult to recognize this way of using
R; values for calculation of bacterial production. The only way to obtain realistic values can be seen in its intercalibration. Because the use of the FDC
method has been disproved for bottom sediments, the dark 14COZ uptake
method in its modern version described above constitutes the only realistic
alternative. The principle of intercalibration is the same as described above for
water. The conversion factor K f between R; and bacterial production (Pb),
expressed in units of mg C cpm- I cm- 3 h- I is calculated as a result of estimation
of both values in parallel experiments. Then the R; values measured as cpm
1 cm- 3 h- I during routine experiments with CH)-TDR can be converted into
units of bacterial production: Pb = R;K f 24 g C cm- 3 day-I.
4.4.4 The Labeled P04P - Uptake Method
The possibility of using the inorganic phosphate uptake rate to estimate microbial growth was mentioned by Karl (1982) and was practically realized by Karl
and Bossard (1985), who approached the calculation of the specific growth
rate of microbial popUlations by estimating the turnover rate of the nucleotide
pool in this way. Moriarty (1990) proposed measuring microbial production in
sediments via estimation of phospholipid synthesis with the aid of labeled
phosphate 3Zp04-p' The labeled phosphate which is added into the natural
water samples is readily consumed by bacteria and incorporated into the most
important constituents of their cells such as nucleic acids, ATP, and phospholipids. I used to measure bacterioplankton production with the aid of 33p04_p
simply by estimating total uptake of P04-P by natural microbial popUlations
separated from the phytoplankton by a special filtration procedure. This
approach was based upon the assumptions that: (1) the internal pool of P04P in the cells of bacterioplankton is negligible in comparison with the external one, and (2) that the use of external P04-P in bacterioplankton is