Leucine incorporation into protein is measured by following the appearance of radioactivity into material that is insoluble in hot trichloracetic acid (TCA). This precipitate is mainly protein and radioactive Leu is essentially associated with only protein (Kirchman et al. 1985), although other macromolecules are also insoluble in hot TCA. In addition, Leu is not transformed to other amino acids, which would also be incorporated into protein and would lead to overestimates of the production rate. Finally, leucine comprises a fairly constant fraction of bacterial protein (Kirchman et al. 1985; Simon and Azam 1989), which implies that changes in leucine incorporation are not due to changes in the leucine/protein ratio.

2.0 Definition
Bacterial production is the rate of synthesis of biomass by heterotrophic bacterioplankton, as estimated by the incorporation of ³H-leucine into the cold trichloroacetic acid-insoluble and ethanol-insoluble cell fraction following a short-term incubation, using a suitable conversion factor, F:
        Bacterial production (cells kg ^-1h ^-1 ) = F*[³ H-leucine] pmole kg ^-1 h ^-1
        F=production of bacterial cells/mole ³ H-leucine

3.0 Principle of analysis
The rate of bacterial production is estimated by tracing the specific incorporation of ³ H-leucine into the TCA-insoluble macromolecular fraction. The incubation is terminated, followed by an extraction of the unincorporated 3H-leucine from the bacterial cells in cold TCA and ethanol.

4.0 Apparatus
4.1 Filtration apparatus. The tritiated incubation solution can be filtered using any reliable, leak-free, acid-resistant multiplace filtration unit.

4.2 Heating block or water bath, 80°C.

4.3 Liquid scintillation analyzer. Samples in liquid scintillation cocktail are counted on a liquid scintillation analyzer, using the following energy window settings:
        Channel a: 0-19 KeV
        Channel B: 2-19 KeV
Samples should be counted long enough to reduce the counting error to < 5-10%.

4.4 Quench corrections. As in previous chapter.

5.0 Reagents.
5.1 Stock of [4,5-3H]-leucine, 40-60 Ci/mmol (New England Nuclear N NET-135H) is stored in the refrigerator and should not be frozen.

5.2 Nonradioactive L-leucine (Sigma L 8000) for making up working solutions.

5.3 Working solution.

5.4 Acid cleaning solution (1N HCl Baker Analyzed) is prepared using Milli-Q water.

5.5 Incubation bottles. As in previous chapter.

5.6 Trichloroacetic acid (TCA) 50% wt:vol.

5.7 Ethanol (80% vol:vol).

5.8 Ethyl acetate (Purified, Baker Analyzed).

5.9 Scintillation cocktail (Packard Ultima-Gold).

5.10 0.45 fm X 25 mm filters, cellulose nitrate or mixed esters of cellulose filters (Milli-pore HAWP 025 00)

6.0 Sampling and incubation.
6.1 Sample water using “clean techniques” (Fuhrman and Bell, 1985). Use plastic gloves to avoid contact with sample. Handling can add amino acids. Acid-rinse sample containers before use. Start incubations as soon as possible (within minutes) after water is sampled.

6.2 Place sample into appropriate incubation containers (two to three replicates) and add ³ H-Leu (final concentration 10 nM). Set up killed control by adding TCA (5% final concentration) to a sample. The sample volume will depend on the environment. For eutrophic environments, 5 or 10 ml will be sufficient. For oligotrophic environments or other environments with low rates, 25 ml may be necessary.

6.3 Incubate from 10 min to 10 h, depending on sample.

6.4 After incubation, add enough 50% TCA to obtain 5% TCA, final concentration. This kills the incubation and starts the extraction.

7.0 Procedures.
7.1 Heat sample to 80°C for 15 min.

7.2 After it has cooled, filter sample through 0.22 or 0.45 mm cellulose filters (e.g. Sartorius cellulose nitrate to be consistent with the thymidine measurements). The vacuum is not critical but should not exceed 150 mm of Hg.

7.3 Rinse filters twice (3 ml) with cold 5% TCA. Rinse twice (2 ml) with cold 80% ethanol (Wicks and Robarts 1988). Remove filter towers and gently rinse (1 ml) with 80% cold ethanol.

7.4 When dry, place filters in scintillation vials. Add 0.5 ml of ethyl acetate to dissolve filter. Filter must be completely at the bottom so that this volume of ethyl acetate is effective. After the filter is dissolved, add filters to appropriate scintillation cocktail and radioassay.

8.0 Calculation and expression of results.
8.1 Theoretical Approach. This approach is called “theoretical” because it is based on literature values of the various parameters needed to relate Leu incorporation to biomass production. Some of these parameters have been measured for samples from natural aquatic environments (Kirchman et al. 1985; Simon and Azam 1989). The equation for relating Leu incorporation to biomass production gC l ^-1 h ^-1 is:
        Production = Leu* 131.2 * (% Leu)-1 * (C/Protein) * ID
where Leu is the rate of Leu incorporation (moles per liter per hour). The other parameters are as follows, with the best, current estimates provided by Simon and Azam (1989):
 

When these best estimates are used, the resulting conversion factor is 3.1 kgC mol ^-1 which is multiplied times the Leu incorporation rate to obtain rates of bacterial biomass production.

8.2 Empirical Approach: The other approach to relate Leu incorporation to bacterial production is the empirical approach which is described in Kirchman and Ducklow (1993). This procedure is used to estimate a conversion factor (cells or gC per mole of Leu incorporated) that converts Leu incorporation into biomass production. This empirical factor, in theory, includes all possible relationships between Leu incorporation
and biomass production, and thus should not be “corrected” further by other factors.

9.0 Other Remarks
9.1 The goal of the Leu method is not to obtain turnover rates of amino acids at in situ concentrations. The added concentration of Leu is purposely much higher than the in situ concentration (usually < 1 nM). Also, organic contamination (unless extremely severe) will not change short-term rates (Kirchman 1990). Contamination by amino acids and other compounds is potentially a serious problem and obviously should be
avoided.

9.2 Two processes can contribute to variations in Leu incorporation that are independent of net biomass production and possibly may lead to errors in estimating bacterial production. First, Leu can be synthesized from other compounds, which leads to isotope dilution of the added radiolabelled Leu. The problem is minimized by adding Leu to concentrations high enough (e.g. 10 nM for marine waters and oligotrophic
lakes) to “swamp” unlabeled Leu and to repress de novo synthesis of intracellular Leu. Isotope dilution experiments can help in selecting the proper concentration (Moriarty and Pollard 1981), although this approach apparently does not guarantee that isotope dilution will be zero (Kirchman et al. 1986; Ellenbroek and Cappenberg 1991). Simon and Azam (1989) directly measured intracellular isotope dilution
using OPA-HPLC and found that it was about 2-fold when 10 nM Leu was added to coastal waters of southern California. Estimates of intracellular isotope dilution can be very useful, but the methodology is difficult and depends on a reasonable separation of phytoplankton from bacteria (Simon and Azam 1989). Addition of higher Leu concentrations should be avoided because some of the radiolabel may diffuse into or may be taken up by microorganisms other than bacteria, e.g. phytoplankton.

The added concentration of ³ H-Leu should be tested in separate experiments (Moriarty and Pollard 1981). For many environments 10 nM of added Leu has proven to be adequate, although much higher concentrations may be necessary in some eutrophic lakes (R. Bell, pers. comm.). If 10 nM is used, it is not necessary that the entire added Leu be radioactive. Leu incorporation is usually high enough such that rate can be measured with a mixture of 0.5 to 1.0 nM 3H-Leu plus 9 to 9.5 nM non-radioactive Leu. This mixture is also quite inexpensive. Rates using this mixture should be corrected for the addition of nonradioactive Leu with the following equation: corrected rate = rate with mixture * (nonradioactive + 3H-Leu)/3H-Leu.

Note only 3H-Leu, without any nonradioactive Leu, should be used in environments where rates are expected to be low, e.g. deep oceans and highly oligotrophic lakes.

9.3 The other potential problem with the Leu method is protein turnover. Microbial cells can synthesize and degrade some proteins, i.e. protein turnover, independent of net growth. Kirchman et al. (1986) found that protein turnover was not important in the only published experiments with natural waters, but protein turnover cannot be ignored, especially when bacterial growth rates are low. If protein turnover is important, Leu incorporation would tend to overestimate biomass production because the radiolabel would be incorporated into new proteins while little radioactivity would be lost as old proteins are degraded. Kirchman et al. (1986) argued that it may be useful to measure protein turnover if organic matter is mineralized during protein
turnover. Even so, it complicates interpretation of Leu incorporation.

9.4 Formalin can also be used for killed controls as abiotic adsorption of radiolabelled Leu in formalin-killed controls is the same as that with TCA. The problem with formalin is that any surface in contact with it should not be used in incubations with live samples. The fumes from formalin are also noxious and could affect live samples.

9.5 Hot TCA extractions of large volumes (> 10 ml) is inconvenient. Alternatively, one can extract the material collected on filters after killing the incubation with a low TCA concentration (0.5%). That is, after filtering the killed sample, the filter is then placed in 5 ml 5% TCA and heated. After extraction and cooling, the 5% TCA is filtered and rinsed. Both filters are radioassayed.

9.6 Because nearly all Leu assimilated is incorporated directly into protein (Kirchman et al. 1985), a simpler TCA extraction is often possible (Chin-Leo and Kirchman 1988). Instead of the hot extraction, the sample is killed with TCA and then filtered (cellulose acetate filters) without the 80 °C extraction. The filter is then rinsed as described above.

10.0 References
Chin-Leo, G. and D.L. Kirchman. 1988. Estimating bacterial production in marine waters from the simultaneous incorporation of thymidine and leucine. Appl. Environ. Microbiol. 54: 1934-1939.
Ellenbroek, F.M. and T.E. Cappenberg. 1991. DNA synthesis and tritiated thymidine incorporation by heterotrophic freshwater bacteria in continuous culture. Appl. Environ. Microbiol. 57: 1675-1682.
Fuhrman, J.A. and T.M. Bell. 1985. Biological considerations in the measurement of dissolved free amino acids in seawater and implications for chemical and microbiological studies. Mar. Ecol. Prog. Ser. 25: 13-21.
Kirchman, D.L. 1990. Limitation of bacterial growth by dissolved organic matter in the subarctic Pacific. Mar. Ecol. Prog. Ser. 62: 47-54.
Kirchman, D.L., E. K’nees and R.E. Hodson. 1985. Leucine incorporation and its potential as a measure of protein synthesis by bacteria in natural aquatic systems. Appl. Environ. Microbiol. 49: 599-607.
Kirchman, D.L., S.Y. Newell and R.E. Hodson. 1986. Incorporation versus biosynthesis of leucine: implications for measuring rates of protein synthesis and biomass production by bacteria in marine systems. Mar. Ecol. Prog. Ser. 32: 47-59.
Moriarty, D.J.W. and P.C. Pollard. 1981. DNA synthesis as a measure of bacterial productivity in seagrass sediments. Mar. Ecol. Prog. Ser. 5: 151-156.
Simon, M. and F. Azam. 1989. Protein content and protein synthesis rates of planktonic marine bacteria. Mar. Ecol. Prog. Ser. 51: 201-213.
Wicks, R.J. and R.D. Robarts. 1988. Ethanol extraction requirements for purification of protein labeled with [ 3 H]leucine in aquatic bacterial production studies. Appl. Environ. Microbiol. 54: 3191-3193.