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12 Measuring Bacterial Biomass
Production and Growth Rates
from Leucine Incorporation in
Natural Aquatic Environments
David Kirchman
College o( Marine Studies, University of Deloware, Lewes,DE 19958, USA
CONTENTS
Introduction
Measuring3H-leucine incorporation by the filter method
Measuring3H-leucine incorporation by the microcentrifuge method
Conclusions
~,~I,~,~,4,~, I N T R O D U C T I O N
The rate of biomass production is a fundamental property of all organisms in nature, but it is an especially important parameter of microbes in
natural aquatic environments. An estimate of microbial production can be
used as a general index of microbial activity and specifically to calculate
growth rates. Since m a n y processes scale with it, biomass production can
be used to obtain a first-order estimate of rates of several processes mediated by microbes. For example, in the case of heterotrophic bacteria, the
organisms to be discussed here, biomass production can be used to estimate use of dissolved organic material (DOM) if coupled with an estimate
of the growth efficiency. That is, DOM uptake equals bacterial biomass
production divided by the growth efficiency (expressed as a fraction, not
a percentage).
Biomass production is the increase in biomass per unit time per unit
w)lume or per area and is a function of both biomass (B), usually
expressed as carbon mass per volume (e.g. lagC l~), and the specific
growth rate (ia) (e.g. h ') (Ducklow, 2000). in the absence of any mortality
(grazers and viruses), bacterial biomass increases exponentially (although
not necessarily very quickly) which can be described by
dB/dt
METt tODS IN MICROBIOLOGY, VOLUME 30
ISBN 0 12 521530-4
= ia B
(12.1)
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In this case, bacterial production is the first derivative ( d B / d t ) or the slope
of the curve on graphs of biomass (B) versus (t). If the data are graphed as
ln(B) versus time, then the slope of this semi-log graph is the growth rate
(V).
In nature, bacteria seldom if ever live in the absence of protist grazers
and viruses, both of which cause bacterial mortality. In most natural
systems, most of the time, bacterial production is matched by mortality
such that (dB/dt),,~.~ = 0. However, the methods discussed here measure
'gross production', i.e. biomass production unaffected by mortality. It is
the rate of biomass production that would occur if mortality were zero.
This is possible because the methods rely on incubations that are relatively short (hour or less) compared to the timescale of bacterial growth
and mortality (a day or longer). Although we say we measure 'gross
production', the rate does not include respiration. Ducklow (2000)
discusses the differences in uses of the terms 'gross' and 'net' production
by bacterial and phytoplankton ecologists.
Ducklow (2000) also reviews the m a n y methods that have been used to
examine bacterial production in aquatic environments. Although alternative methods are still valuable for specific applications, recent investigations of natural aquatic environments have used either thymidine (TdR)
incorporation or leucine (Leu) incorporation or both to estimate bacterial
production. The two methods, which were originally proposed by
Fuhrman and Azam (1980) and Kirchman et al. (1985), respectively, have
many parallels, and the experimental details are nearly identical. Both are
rapid, easy, and specific for heterotrophic bacteria, some of the main
reasons w h y they have been widely adapted by microbial ecologists. I
focus here on the leucine method because it is more straightforward than
the TdR method to estimate bacterial production; if one relies on 'theoretical' conversion factors, the only variable assumption in extrapolating
from Leu incorporation to bacterial production is the degree of isotope
dilution (Kirchman, 1993), as described below. Consequently, over the last
few years more investigators seem to be using Leu incorporation rather
than the TdR method.
M E A S U R I N G 3H-LEUCINE I N C O R P O R A T I O N
B Y T H E FILTER M E T H O D
Principle
Two variations of the Leu method will be described, but the basic
biochemistry and physiology behind the methods are the same. Leucine
incorporation is often used to measure protein synthesis in pure cultures
of bacteria because leucine is a constant proportion of all protein (7.3~7~;
Kirchman at al., 1985). Consequently, rates of protein synthesis can be estimated from the rate at which this amino acid appears in the protein fraction. The original method called for hot trichloroacetic acid (TCA)
extraction to measure leucine incorporated specifically into protein, but
this step is not necessary because the difference between a cold and hot
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TCA extraction is negligible; nearly all leucine is incorporated directly
into protein (Kirchman et al., 1985). Rates of total biomass production can
be estimated in turn if the a m o u n t of protein per cell or per cellular mass
is known. Although cell size and thus protein per celt can vary greatly,
protein is a relatively constant fraction of bacterial biomass (60% of dry
weight; Simon and Azam, 1989). Incorporation of leucine (and thymidine)
is dominated by heterotrophic bacteria in most aquatic habitats.
Rates of leucine incorporation into protein are estimated from the
appearance of radioactivity, added as 'H-leucine, in the protein fraction.
The added ~H-leucine (20 nM) normally is much higher than in situ concentrations (< 1 nM). This high added concentration has two effects. First,
it means that the natural extracellular leucine usually can be ignored in all
calculations. Second, because extracellular concentrations are so high with
the added leucine, bacteria will take up the exogenous ~H-leucine and
repress leucine biosynthesis, i.e. the production of non-radioactive leucine
which is subsequently used for protein synthesis. But the problem is, some
a m o u n t of leucine biosynthesis, usually unknown, may continue even in
the presence of high exogenous leucine, thus 'diluting' incorporation of
radioactive leucine. This is called 'isotope dilution' (ID).
In summary, the equation describing biomass production as estimated
from leucine incorporation (Leu incorp) is:
Biomass production = Leu Incorp x 131.2 + (Leu per protein)
x (cell C per protein) x ID
(12.2)
which gives biomass production as gC per volume per unit time of the
incubation. The molecular weight of leucine is 131.2 and converts moles of
leucine incorporation into grams of C. The fraction of Leu in protein ('Leu
per protein') is 7.3% (0.073 in the equation) and cellular C per protein is
0.86 (Simon and Azam, 1989). The few measured estimates of ID average
to about 2 (Simon and Azam, 1989). A more conservative approach (lower
production) would assume that ID = 1. In which case, bacterial production
can be calculated as:
Biomass production = Leu lncorp x 1.5 kg C per mol
(12.3)
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when leucine incorporation is measured in moles incorporated per unit
time and volume.
Specific growth rates can be estimated from rates of biomass production if a few assumptions are made. If we apply Equation (12.1) to gross
production (not net production), i.e. that measured by the TdR or Leu
approaches, then the specific growth rate (~1) is:
p - BP/B
(12.4)
where B P is biomass production (biomass per time unit per volume or
area) and B is a measure of bacterial abundance (cells per volume) or
biomass (cellular mass per volume). Cellular biomass is usually estimated
by multiplying bacterial abundance by a carbon per cell conversion factor,
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e.g. 10 fgC per cell (Fukuda et al., 1998). Biomass versus cell production is
discussed below.
Note that growth rates have units of 'per time', e.g. d 1, whereas generation time (g) is
g = ln(2)/p = 0.693/p
(12.5)
and has units of 'time', e.g. hours or days. Ecologists often discuss
'doublings per day' which has the same units as specific growth rates, but
is calculated as 1/g.
The growth rate calculated from biomass production would reflect an
average for the entire bacterial assemblage, including both the very slow
and the very fast growing cells. The extreme is the case of nonviable cells
with a growth rate of zero. Over the years, there has been much debate
about the fraction of inactive (if not dead) bacterial cells in aquatic habitats, but generally a high proportion of cells do incorporate leucine
(Kirchman et al., 1985) and thymidine (Fuhrman and Azam, 1982).
However, regardless of the capacity of bacteria to incorporate these two
compounds, several studies do suggest that activity and presumably
growth rates vary greatly among various members of the bacterial assemblages (del Giorgio et al., 1997; Sherr et al., 1999). Although the limitations
are obvious, growth rates calculated by the method outlined above can be
very useful in characterizing bacterial assemblages. Also, there are few
alternative approaches.
Equipment and reagents
• Tritiated leucine ([2,3,4-3H] leucine) as a stock solution with specific
activity of >60 nmol per Ci (I mCi ml -~) (NEN,Amersham).
• Polycarbonate incubation tubes or flasks of appropriate sizes for environment. Other materials (e.g. glass or other plastics) should be avoided.
• Vacuum pump, flasks and filter holders (25 mm) for filtering radioactive
and corrosive liquids.
• Filters of mixed cellulose esters with pore sizes of 0.45 pm (or 0.22 lum)
and diameter of 25 mm (Millipore).Two pairs of forceps. It may be necessary to remove the plastic handle so that the forceps can fit down to the
bottom of a 7 ml scintillation vial.
• Pipettes (e.g. Pipetman) that dispense volumes ranging from microliters
(for the 3H-leucine) to milliliters. Repeating dispensers for the ethyl
acetate and scintillation cocktail.
• Trichloroacetic acid (TCA) in a stock solution of 50% and as a wash solution (5%)
• Ethanol, 80%.
• Ethyl acetate.
• Scintillation cocktail and scintillation vials (7 ml). Ultima-Gold (Packard
Instruments) was found to be the optimal cocktail (Ducklow, personal
communication).
230
•
•
•
Vortexer.
Scintillation counter (e.g. Beckman,Wallac).
Appropriate containers for radioactive corrosive liquids and radioactive
solids.
Assay
It is not necessary to use sterile techniques to conduct the following
assay. Contamination by organic material, however, should be avoided
as it could stimulate bacterial growth or dilute the added 3H-leucine.
Plastic gloves should be worn at all times to protect the sample from
contamination.
1. Add an appropriate volume of the water sample to an incubation
vessel. The volume may range from 5 ml of highly active waters to
100 ml of very inactive waters. Duplicates or triplicates should be
prepared per sample.
2. Prepare a killed control by adding TCA to a final concentration of
1%. Other killing agents can be used (e.g. formaldehyde), but TCA
is needed for other steps in the assay anyway and it does not affect
the incubation vessel. Bottles exposed to formaldehyde or gluteraldehyde should not be used for live incubations. Also, formaldehyde and gluteraldehyde need to be handled in a hood.
3. Add 3H-leucine to the samples and to the killed control and then
mix by hand. The final concentration of leucine should be 20 riM,
which usually is sufficient to maximize incorporation rates, indicating that isotope dilution has been minimized. This maximum
concentration can be determined empirically by simply varying
the added 3H-leucine concentration and noting at what concentration 3H-leucine incorporation reaches a maximum. The actual
volume of ~H-leucine added per incubation will vary, depending
on the incubation volume and the specific activity of the 3Hleucine batch from the manufacturer.
4. Incubate the samples at the in situ temperature for an appropriate
time. The incubation time may vary from 30min to 24h,
depending on the activity level. For many applications, end point
determinations are quite adequate, but for unknown environments it is advisable to measure incorporation over time in order
to determine the best single incubation time. The best time is the
shortest period that gives a measurable rate with acceptable errors.
For most water samples, this time will be about one hour. Water
samples without visible particles probably do not need to be
shaken during the incubation, but if material is present that may
settle, it is advisable to shake gently.
5. After incubation, filter samples through filters using minimal
vacuum (<200 mm Hg). The start and end times of the incubation
are noted.
6. With the vacuum connected, rinse the filter twice with 3 ml of icecold TCA.
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7. Rinse twice with 3 ml of ice-cold 80% ethanol.
8. With the vacuum still connected, lift the tower off the filter and use
a small volume of ethanol to rinse the filter edge that was covered
by the tower.
9. Using two forceps, fold the filter into quarters and place it at the
bottom of the 7 ml scintillation vial. It is important to force the
filter to the bottom of the vial so that the small volume of ethyl
acetate (step 11) will completely cover the filter.
10. Allow the filter to dry before proceeding to the next step. Any
ethanol remaining on the filter must be evaporated as it is a potent
quencher in scintillation counting.
11. Add 0.5 ml of ethyl acetate to the scintillation vial.
12. Once the filter is dissolved, add 5 ml of scintillation cocktail, and
vortex the vial briefly.
13. It is usually necessary to allow the sample to sit for as long as two
days to maximize the dispersion of the radioactivity into the cocktail. Vortex briefly before scintillation counting.
Rate of leucine incorporation (Leu incorp as nmol 1 ~h ') is calculated as
follows:
Leu incorp = {(dpm on sample filter)-(dpm in killed control)}/
incubation time/(2.22 × 10" d p m per tJCi) x (Leu specific activity as
nmol per laCi)
The factor 2.22 x 10" d p m per laCi converts the radioactivity (dpms)
found on the filter to laCi, the unit of radioactivity used for the leucine
specific activity. This specific activity is provided for each batch of
leucine by the manufacturer and converts radioactivity (tJCi) to moles
of leucine incorporated.
M E A S U R I N G 3H-LEUCINE I N C O R P O R A T I O N
BYTHE MICROCENTRIFUGE METHOD
Principle
The following procedure, which was originally proposed by Smith and
Azam (1992), is nearly the same as the flter method just described. The
critical differences are that the incubation and radioassaying are both
done in a 2.0 ml microcentrifuge tube. Also, the radioactivity incorporated
into the microbial biomass is collected by centrifugation, not filtration. As
a result, the amount of radioactivity and the volume of sample and scintillation cocktail are much smaller than used in the filter method. Also, the
processing time and variability among replicates are often better with the
microcentrifuge method. Finall),, the 'blank' or radioactivity in the killed
controls is usually much lower for the microcentrifuge method (especially
when TdR incorporation is measured), which allows much lower rates to
be measured in shorter incubation times. This is especially important
w h e n samples from deep waters with low activity are studied.
232
The m e t h o d described below has been extensively used in seawater
and to a lesser extent in freshwaters. For freshwater systems, precipitation
m a y be aided by adding humics (Kirschner and Velimirox; 1999) or NaC1
(to 3.5'7,, final concentration; D.C. Smith, personal communication), but
other investigators do not add anything (J.J. Cole, personal communication). The need for additions to aid in precipitation should be assessed for
each freshwater system u n d e r study. Additions are not needed for
seawater studies.
Equipment and reagents
• Tritiated leucine ([2,3,4-3H] leucine) as a stock solution with specific
activity >60 nmol per Ci (I mCi ml ') (NEN, Amersham).
• 2 ml microcentrifuge tubes.
• Pipettes (e.g. Pipetman) that dispense volumes ranging from microliters
(for the 3H-leucine) to milliliters. Repeating dispensers for the washes and
scintillation cocktail.
• Trichloroacetic acid (TCA) in a concentrated solution (100% w/v) and as a
wash solution (5%).
• Ethanol, 80%.
• Aspirator, which can be constructed with tubing and a vacuum pump.
• Scintillation cocktail (7 ml). Ultima-Gold (Packard Instruments) was found
to be the optimal cocktail (H.W. Ducklow, personal communication).
• Vortexer.
• Microcentrifuge (e.g. Eppendorf).
• 7 ml plastic scintillation vials (can be re-used) as carriers for the microcentrifuge tube. Scintillation vials from some manufacturers are smaller
than others which makes it difficult to remove the microcentrifuge tube.
• Scintillation counter (e.g. Beckman,Wallac).
• Appropriate containers for radioactive corrosive liquids and radioactive
solids.
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Assay
°n
The same general comments about sterile technique, contamination,
and gloves that apply to the filter m e t h o d also apply to the microcentrifuge method.
1. Add an appropriate v o l u m e of ['H] leucine to each microfuge tube
before the sample is added. The tubes need not be washed before
use. The stock [~H] leucine will need to be diluted such that a small
but reasonable v o l u m e (between 2 and 5 Ill) of [:'HI leucine can be
a d d e d to each tube. As with the filter method, the final concentration of [~H] leucine should be 20 nM, but this can be tested as
described above. Killed controls are prepared by adding 89 pl of
100~ TCA to selected tubes. It is convenient to mark one side of
the tube (cap and top edge) for positioning in the microcentrifuge
(see step 7).
233
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2. Add sample water (1.7 ml) to each microfuge tube and shake the
tube by inverting it. Duplicates or triplicates should be prepared
per sample.
3. Incubate the samples at the i~l sihl temperature for an appropriate
time period as described for the filter method.
4. After incubation, add 89 t~1of 100% TCA to each sample, except for
the killed controls. (For measuring TdR incorporation, the samples
must be cooled to 4°C by placing the tubes in ice before addition of
the TCA.)
5. Place the tubes in the microcentrifuge with the mark on the
outside, i.e. the side where the pellet will eventually form.
Centrifuge the samples at the maximum speed of the microcentrifuge for 10 min.
6. After centrifugation, remove the supernatant by aspiration, being
careful to avoid the pellet which is on the marked side.
7. Add l ml of ice-cold TCA to each tube, vortex, and repeat the
centrifugation step.
8. Remove the TCA by aspiration as described in step 7 and add
1.0 ml of ice-cold 80% ethanol. Centrifuge again.
9. Remove the ethanol by aspiration.
10. Allow the pellet to dry completely because any remaining ethanol
will cause quenching during liquid scintillation counting and lead
to erratic results.
11. Add 1 ml of scintillation cocktail to the microcentrifuge tube and
then vortex briefly.
12. Place the microcentrifuge tubes in plastic 7 ml scintillation vials
and radioassay.
13. It is usually necessary to allow the sample to sit for as long as two
days to maximize the dispersion of the radioactivity into the cocktail. Vortex briefly before scintillation counting.
Rates of leucine incorporation (Leu incorp as nmol 1 ' h 1)are calculated
using the same equation as used for the filter method.
CONCLUSIONS
ideally both leucine and thymidine methods should be used because they
provide independent estimates of bacterial production. The dual label
method with I4C-leucine and ~H-thymidine allows both incorporation
rates to be estimated in a single incubation tube (Chin-Leo and Kirchman,
1988). Some investigators have used leucine as a measure of biomass
production (gC per liter per day) and thymidine for cell production (cells
per liter per day). Their rationale is that leucine measures protein
synthesis, i.e. biomass production, whereas thymidine reflects DNA
synthesis, i.e. cell production. During balanced growth, however, the two
rates have to be equal (when converted to equivalent units) because for
sustained periods, cells cannot increase mass without division nor can
234
they divide without making new biomass. For this reason, leucine and
thymidine incorporation rates cannot diverge for long time periods
(greater than a couple of generation times or roughly a few days) or over
large geographic areas. In practice, leucine and thymidine incorporation
rates usually covary and are highly correlated. But since bacterial growth
is not necessarily balanced, rates of leucine and thymidine incorporation
may diverge and not be correlated. The difference between leucine and
thymidine incorporation may be informative, if coupled with other
measurements of the biogeochemical environment.
Rates of leucine and thymidine incorporation could also diverge
because the relationship between incorporation rates and bacterial production, which is reflected in conversion factors, may change. For
example, changes in DNA content per cell, perhaps due to changes in bacterial species composition, would lead to variation in thymidine incorporation without changes in the actual rate of biomass production. Changes
in isotope dilution is probably the biggest unknown in calculating production from leucine incorporation. The problem of picking the correct
conversion factor is the difficult part of using either leucine or thymidine
incorporation as a measure of bacterial production. Using 'empirical' conversion factors potentially is a solution, but it is far from perfect. Ducklow
(2000) provides the most recent review of these questions.
It should be emphasized that often rates of leucine and thymidine
incorporation alone, e.g. moles of leucine incorporated per liter per day,
are sufficient for addressing ecological questions. Similarly, often an estimate of incorporation rates per cell is an adequate index of bacterial
growth rates. Even when using just incorporation rates, however, one
conceptional problem with the leucine method should be mentioned.
Unlike DNA, protein can 'turn over', i.e. protein is degraded within a
cell and new protein is synthesized, in prokaryotes protein turnover is
generally thought to be negligible, but conceivably it is substantial
compared to total protein synthesis when cells are growing very slowly,
i.e. the case with some aquatic environments. If protein turnover is
substantial, then leucine incorporation would overestimate bacterial
production. In the extreme case of high protein turnover and zero net
protein synthesis (and thus zero biomass production), PHlleucine would
be incorporated into the new protein but no radioactivity would be lost
during the degradation of the old (non-radioactive) protein. Thus, the
leucine method would indicate some positive rate of biomass production
when in fact there was none. The single study of protein turnover in an
aquatic environment (Kirchman et al., 1986) did not find substantial rates,
but more work is needed on this question.
The other main conceptual problem with both the leucine and thymidine method is that it measures total production of the entire community.
It is now well recognized that the heterotrophic bacterial community is
very diverse and that the role in biogeochemical cycles for each member
of this community is likely to differ. For this reason, there is a need to
develop methods for measuring biomass production and growth rates for
specific members or groups within the bacterial community. Some
progress has been made (Kemp et al., 1993; Urbach et al., 1999), but more
235
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w o r k is n e e d e d . It is conceivable that bacterial p r o d u c t i o n will be m o r e
accurately estimated b y a d d i n g u p the c o n t r i b u t i o n s b y the m a j o r bacterial g r o u p s rather t h a n t r y i n g to m e a s u r e a c o m m u n i t y rate, as
described here.
References
Chin-Leo, G. and Kirchman, D. L. (1988). Estimating bacterial production in
marine waters from the simultaneous incorporation of thymidine and leucine.
Appl. Environ. Microbiol. 54, 1934-1939.
del Giorgio, P. A., Prairie, Y. T. and Bird, D. E (1997). Coupling between rates of
bacterial production and the abundance of metabolically active bacteria in lakes,
enumerated using CTC reduction and flow cytometry. Microbial Ecol. 34, 144-154.
Ducklow, H. W. (2000). Bacterial production and biomass in the oceans. In:
Microbial Ecology o(tlle Oceans (D. L. Kirchman, Ed.). John Wiley and Sons, New
York.
Fuhrman, J. A. and Azam, F. (1980). Bacterioplankton secondary production estimates for coastal waters of British Columbia, Antarctica, and California. Appl.
Environ. Microbiol. 39, 1085-1095.
Euhrman, J. A. and Azam, E (1982). Thymidine incorporation as a measure of
heterotrophic bacterioplankton production in marine surface waters: evaluation
and field results. Mar. Biol. 66, 109-120.
Fukuda, R., Ogawa, H., Nagata, T. and Koike, 1. (1998). Direct determination of
carbon and nitrogen contents of natural bacterial assemblages in marine environments. Appl. Environ. Microbiol. 64, 3352-3358.
Kemp, P. E, Lee, S. and LaRoche, J. (1993). Estimating the growth rate of slowly
growing marine bacteria from RNA content. Appl. Envi~vn. Microbiol. 59,
2594-2601.
Kirchman, D. L. (1993). Leucine incorporation as a measure of biomass production
by heterotrophic bacteria. In: Current Methods in Aquatic Microbial Ecology (P.
Kemp, B. E Sherr, E. B. Sherr and J. J. Cole, Eds). Lewis Publishers, Boca Raton,
EL.
Kirchman, D. L., K'nees, E. and Hodson, R. E. (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., Newell, S. Y. and Hodson, R. E. (1986). Incorporation versus biosynthesis of leucine: implications for measuring rates of protein synthesis and
biomass production by bacteria in marine systems. Mar. Ecol. Prog. Set. 32, 47-59.
Kirschner, A. K. T. and Velimirov, B. (1999). Modification of the H-3-1eucine
centrifugation method for determining bacterial protein synthesis in freshwater
samples. Aquatic Microbial Ecol. 17, 201-206.
Sherr, B. E, del Giorgio, P. and Sherr, E. B. (1999). Estimating abundance and
single-cell characteristics of respiring bacteria via the redox dye CTC. Aquatic
Microbial Ecol. 18, 117-131.
Simon, M. and Azam, E (1989). Protein content and protein synthesis rates of
planktonic marine bacteria. Mar. Ecol. Prog. Set. 51, 201-213.
Smith, D. C. and Azam, E (1992). A sirnple, economical method for measuring
bacterial protein synthesis in seawater using ~H-leucine. Mar. Micn)b. Food Webs
6, 107-114.
Urbach, E., Vergin, K. L. and Giovannoni, S. J. (1999). hnmunochemical detection
and isolation of DNA from metabolically active bacteria. Appl. Environ.
Microbiol. 65, 1207-1213.
236
List of suppliers
The following is a selection of companies. For most products, alternative
suppliers are available.
Amersham Life Science, Inc.
26111 Miles Road
Cleveland, OH 44128, USA
Teh 1-216-765-5000
Fax: 1-216-464-5075
http://www.amersham.co.uk
Radiochemicals ([~H]Leu,
[~H]TdR).
Packard Instrument Company
800 Research Parkway
Meriden, CT 06450, USA
Teh 1-800-323-1891 USA Only
1-203-238-2351
Fax: 1-203-639-2172
E-maih [email protected]
Scintillation counter; scintillation
cocktail.
Beckman Instruments, Inc.
2500 Harbor Blvd.
Fullerton, CA 92634-3100, USA
Teh 1-714-871-4848
Fax: 1-714-773-8283
1-800-643-4366
Rainin Instrument Co., Inc.
Mack Road, Box 4026
Woburn, MA 01888-4026, USA
Tel: 617-935-3050
Pipetters.
Scintillation counter.
Millipore Corporation
80 Ashby Road
Bedford, MA 01730, USA
Teh 1-800- 645-5476
1-617-275-9200
Fax: (508) 624-8873
Wallac Inc., Berthold
9238 Gaither Road
Gaithersburg, MD 20877, USA
Tel: 1-301-963-3200
1-800-638-6692
Fax: 1-301-963-7780
e-mail: [email protected]
Filters.
Scintillation counter.
NEN Life Science Products
549 Albany Street
Boston, MA 02118, USA
Tel: 1-800-551-2121; 1-617-482-9595
Fax: 1-617-482-1380
Radiochemicals ([~H]Leu, [~H]TdR).
237
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