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HOW TO MEASURE GRAZING ON
HETEROTROPHIC BACTERIA
By
Dolors Vaqué
Department of Marine Biology and Oceanography
Institut de Ciències del Mar de Barcelona
Passeig Marítim de la Barceloneta 37-49
08003 - Barcelona
e-mail: dolors@icm.csic.es
Determination of grazing on bacteria by protozoa is based on the use of
fluorescent labelled bacteria. These are heat-killed bacteria stained with a fluorocrome
(DTAF, FITC…). Hence, they cannot divide, and grazing rates can be measured by
following their disappearance over time. The used FLB is P. diminuta that for its size
(very similar to the natural bacteria) makes it very convenient for this kind of analysis.
Besides knowing and determining how many bacteria are consumed by protozoa, it is
also important to know the abundance and biomass both of main prey (heterotrophic
bacteria) and alternative preys (Synnechoccocus and Proclorophytes), as well as
predators (nanoflagellates and ciliates).
This protocol is addressed to all students and researchers interested in this kind of
methodology. We therefore include the following points:
1
1- OBTENTION, STAINING PROCEDURE AND WORKING SOLUTION OF
P. diminuta
a) OBTENTION
b) PREPARATION AND STAINING
c) WORKING SOLUTION
2- DETERMINATION OF GRAZING ON BACTERIA
a) SAMPLING MANIPULATION
b) DETERMINATION OF VARIABLES
b.1) BACTERIAL PRODUCTION
b.2) ABUNDANCE AND BIOMASS OF PICOPLANKTON BY FLOW
CYTOMETRY
b.3) ABUNDANCE AND BIOMASS OF PICO AND NANOPLANKTON
BY EPIFLUORESCENCE MICROSCOPY
b.4) CALCULATION OF GRAZING RATES, CELLS AND CARBON
CONSUMPTION
b.5) CILIATES ABUNDANCE AND BIOMASS BY INVERTED
MICROSCOPY
ANNEX: NECESSARY MATERIAL FOR:
a) SAMPLING
b) BACTERIAL PRODUCTION
c) FLOW CYTOMETRY ANALYSIS
d) EPIFLUORESCENCE COUNTS
e) CILIATE ABUNDANCE
RELEVANT BIBLIOGRAPHY
ACKNOWLEDGMENTS
2
1- OBTENTION, STAINING PROCEDURE AND WORKING SOLUTION OF
P. diminuta
a) OBTENTION
Culture medium
LB Medium (Luria - Bertrani):
Bacto-tryptone o Peptone 10 g
Bacto yeast Extract
5g
NaCl
10 g
Agar
15 g
Distilled water
1L
pH: 7.5
-Autoclave 20 minutes.
-Leave the medium to cool down and fill the culture plates with the culture medium
under a laminar flow hood.
-Wait until solidification.
Harvest
-Inoculate the P. diminuta strain (in sterile conditions) to the culture plates filled with
LB medium (Maniatis et al. 1982). The P. diminuta strain was obtained from the
Spanish Type Culture Collection (Burjasot, València).
-Leave it to grow at 20 oC (3 or 4 days), and transfer to new plates every other week.
The use of two-week old P. diminuta cultures is recommended to achieve small cells
(approx. 0.065 µm3).
b)STAINING PROCEDURE
-P. diminuta tracers were produced by scraping cells from two-week old agar plates
and suspending them in carbonate-bicarbonate buffer (Na2 CO3-NaHCO3).
-The suspension is made in 10 eppendorfs vials, adding one ml of buffer in each.
Buffer solution
-Na2 CO3 0.5 N (5.3 g/100 ml of distilled water): 1 volume
-Na HCO3 0.5 N (4.2 g/100 ml of distilled water): 3 volumes
3
Example of buffer solution:
20 ml of Na2CO3 0.5 N + 60 ml of NaHCO3 0.5 N.
-Sterilize the solution by filtration (through 0.2 µm filters) before adding to the
eppendorfs vials.
Staining
-To each eppendorf vial containing the P. diminuta solution, add 100 µg ml-1 of
DTAF or FITC (final concentration, diluted previously with the buffer solution).
-Stir each vial with a vortex (1 min.).
-Incubate the cell suspension (with the dye) for 2 hours in a water bath at 60 oC.
-After 2 hours incubation, stir each vial again with the solution of stained cells.
-Rinse the stained cells with filtered seawater (< 0.2 µm) or carbonate-bicarbonate
buffer, ressuspend and centrifuge 3 to 5 times (10 min, 10000 – 12000 rpm, IEC
Micromax centrifuge, 851 rotor) to prevent the transfer of leftover dye to the natural
samples.
-Sonicate all eppendorf vials in an ultrasonic bath (Cole Palmer) for 5 min.
-Pool together the different eppendorf solutions of P. diminuta in a unique 10 ml vial.
This will be the STOCK solution.
c)WORKING SOLUTION OF P. diminuta
-Sonicate the STOCK solution for 5 minutes.
-Take 5 or 10 µl of the STOCK solution and dilute them in 10 ml buffer, previously
filtered through 0.2 µm.
-Sonicate, again, the new solution for 5 minutes and make a quick cells count by
epifluorescence or flow cytometry, to know the exact abundance. In this case it is
better to do it by epifluorescence and see whether P. diminuta cells are properly
dispersed or there are aggregations. If we observe a large number of clumps, we
should sonicate again (THIS STEP IS VERY IMPORTANT).
-The usual concentration for the STOCK solution is approx. of 109-1010 P. diminuta
per ml (this means that in 10 ml we will have around 1010-1011 cells).
-From the STOCK solution (~ 109 cells ml-1) take 0.2 ml and add them to different
vials, and dilute them with buffer to achieve the P. diminuta concentration needed to
use in every grazing determination. This is the WORKING SOLUTION. Keep it
frozen (-20 oC ), until its use.
-Depending on the sampling site, we will have around 5 x 108-109 bacteria per litre.
Then the WORKING SOLUTION of P. diminuta should be around 108-0.2 x 109 P.
diminuta per vial.
4
-THE WORKING SOLUTION of P. diminuta should be around 20-30% of the
natural bacteria concentration (cell L-1).
2- DETERMINATION OF BACTERIAL GRAZING
a)SAMPLING MANIPULATION
-From the sampling site take 3 litres of water sample.
-Pre-filter the 3-litre sample through a 50 µm net to avoid predators higher than
protozoa (nanoflagellates and ciliates). Keep the sample in a plastic carboy.
-Alternatively, do not filter the initial sample if we want to know the grazing impact
on bacteria of the total planktonic community (protozoa, naupliae, filter feeding
zooplankton…)
-Unfreeze and sonicate the working solution of P. diminuta (which from now on we
will call FLB) for 5 minutes. If we have available an epifluorescence microscope it is
recommendable to make a quick observation of the prepared FLB's before adding
them to the sample. We want to test if they are evenly distributed (no aggregations).
-Add the appropriate work solution to the 3-litre sample.
-Gently rotate the carboys (approx. 20 times).
-Fill three 1L bottles (plastic or nalgene) with 1L sample each.
-One of the bottles will be used as a control (killed control: add 100 ml of
glutaraldehyde 10%, or live control: filter one litre of water through 0.8 µm to avoid
predators, and after add an adequate work solution of FLB's). This control will tell us
if the disappearance of FLB's over time is due to predation, or if there are any losses
of FLB due to losses of fluorescence or attachment on the bottle wall.
Incubation time and aliquots sampling
-Each 1L bottle with sample and FLB's (control and duplicates) are incubated for 2448 h at in situ temperature (incubation bath, incubator chamber). Light conditions are
constrained to our requirements; we can incubate at in situ light conditions or in the
dark.
*Samples and aliquots taken from each duplicate at time zero:
i)Take 2 extra litres of water from the same sampling site. Fill two plastic bottles, with
1L each, to determine abundance and cell volume of ciliates by an inverted
microscope.
5
-Samples are fixed with acidic-lugol (1-10% final concentration).
-Keep the fixed samples in the dark at 4 oC.
ii)From each duplicate take aliquots of 1.2 ml (and keep them in criovials) to
determine abundance and cell volume of heterotrophic bacteria, Proclorophytes,
Synnechoccocus, Picoeucaryotes and the added P. diminuta. All these parametres will
be determined by flow cytometre.
-Aliquots are fixed with 0.12 ml of1% paraformaldehyde + 0.05% glutaraldehyde
(final concentration).
-Stir the criovials (aliquots + preservative).
-Keep the criovials in the dark for 10 minutes.
-Freeze the criovials suddenly in liquid nitrogen.
-Once frozen, keep the criovials in a regular freezer (from -20 to -80 oC) until their
analysis.
iii)Take 100 ml aliquots (kept them in plastic bottles) to determine abundance of
nanoflagellates, heterotrophic bacteria, Synnechoccocus and FLB'S by
epifluorescence.
-Aliquots are fixed with 10 ml de glutaraldehyde (1% final concentration).
-Keep the 100 ml in the refrigerator until their filtration. IMPORTANT: filtrate the
aliquots and make slides as soon as possible after sampling in order to optimize the
cells observation by epifluorescence. We recommend to filter the aliquots within the
sampling week.
iii)Take aliquots of 1.2 ml (quadruplicates, 3 samples and 1 killed control) to
determine bacterial production by tritiated leucine incorporation (see Gasol 1999 a).
Aliquots taken at 8-12 h intervals to measure:
Abundance and cell volume of picoplankton (phototrophic and heterotrophic) and P.
diminuta by flow cytometry, as well as bacterial production (volumes and sampling
procedures are the same as for time zero).
*Aliquots taken at the end of incubation time (~ 48 h) to measure:
The same variables as at time zero (except for ciliate abundance, which are taken only
at time zero).
Note: Duplication of picoplankton and FLB's counts (by flow cytometry and
epifluorescence) at the beginning and at the end of the incubation time are made just
to test the results obtained by flow cytometry.
6
b)VARIABLES DETERMINATION
b.1)HETEROTROPHIC BACTERIAL PRODUCTION
Follow the protocol described in Gasol (1999a).
b.2)ABUNDANCE AND BIOMASS OF PICOPLANKTON BY FLOW
CYTOMETRY
-Unfreeze the criovials just before starting the sample processing. IMPORTANT: only
unfreeze the number of samples that we are capable of processing. P. diminuta can
lose fluorescence if we unfreeze a sample and we freeze it again. The way that the
flow cytometre is used to count picoplankton (heterotrophic and phototrophic) is
described in the protocol developed by Gasol (1999b).
-To count the stained (DTAF or FITC) P. diminuta, add 10 µl of beads solution to
0.2-0.4 ml of sample (~ 106 per ml).
-Flow cytometre settings for P. diminuta are: FSC, EO2; SSC, 427; FL1, 551; FL2,
475; FL3, 590; FL4, 413; DDM Param, FL1.
-As for heterotrophic bacteria as well as for FLB's, in the adquisition data plot, first
we observe the appearance of a cloud corresponding to the beads and after a cloud of
FLB's as is indicated in Fig. 1.
-All process of the flow cytometre use and data adquisition are described in Gasol
(1999b).
IMPORTANT:If we want to make duplicates of P. diminuta counting by
epifluorescence and flow cytometry we recommend using DTAF as dye instead of
FITC. Although cells stained with FITC give a good resolution, when counted by
flow cytometre (Fig. 1), these are very difficult to observe by epifluorescence
microscopy.
7
Green fluorescence (FL1)
Green fluorescence (FL1)
n
c
b
n
c
b
B
A
90°light scatter (SSC)
Fig. 1. Adquisition data plots of P. diminuta. FL1 (green fluorescence) in front of 90º
light scatter. (A)Stained-DTAF P. diminuta in sea water samples. (B)Stained-FITC P.
diminuta in seawater samples.
Bacterial abundance calculation
We can evaluate bacterial abundance by following two procedures:
i)From the flux speed (of the flow cytometre) used for each sample (Low, Medium,
High). For P. diminuta we use LOW. IMPORTANT!!!! We need to know how many
seconds every sample takes to achieve 10000 events. The flux speed in µl min-1 from
a calibration curve performed in April 2000 is:
LOW: 27
MEDIUM: 36
HIGH: 66
Abundance of P. diminuta ml-1 is obtained as the fluorescence value for P. diminuta,
divided by the number of seconds (Ts) employed in each sample, and the speed flux
(µl per second). This value depends on the speed flux used (LOW, MEDIUM or
HIGH, previously calibrated). Finally we multiply by 1000 to convert µl to ml, by 60
to convert seconds to minutes and by the dilution factor (dil F.), which come from the
addition of a 10% of volume of the preservative to the sample volume.
P. diminuta ml-1 = FL (P. diminuta)*1000 µl * 60 s* (dil F) / (Ts * µl s-1)
ii)From Beads calibration:
8
P. diminuta ml-1 = [FL (P. diminuta)/(FL (Beads)]*[Beads* µl added*1
ml/1000µl]*1 ml of sample / volume of sample * dil factor of the sample
-Fig. 2 shows an example of the evolution of heterotrophic bacteria and P. diminuta
abundance over time from a sample taken in the Northwestern Mediterranean.
2.4 10 5
Coastal
Surface
DCM
(Palamós)
2 10 6
1.6 10
2.2 10 5
2 10 5
6
1.8 10 5
1.6 10 5
1.2 10 6
1.4 10 5
8 10 5
4 10
1.2 10 5
5
1 10 5
8 10 4
0
10
20
30
40
50
0
10
20
30
40
50
Fig. 2. Left panel: Evolution of heterotrophic bacteria over time. Right panel:
evolution over time of P. diminuta, within the incubation bottles .
-Fig. 3 shows the evolution of P. diminuta abundance over time in a control sample
2 105
P. diminuta (cel ml- 1)
Bacterial Abundance (ml-1 )
2.4 10 6
1.5 105
1 105
5 104
0
10
20
30
40
50
Time (hours)
.
Fig. 3. Evolution of P. diminuta over time in a control sample (without predators).
(•)water filtered through 0.8 µm; (o) water filtered through 0.2 µm. Triplicates
samples (bars = Standard error).
9
Cell volume and biomass calculations
Heterotrophic bacteria cells volume can be obtained from the ratio between FL1
bacteria values and FL1 Beads values and their correspondence with the cellular size
found by image analysis (Gasol, 1999b) following the equation:
µm3 cell-1 = 0.0075 + 0.11* (FL1 bacteria / FL1 Beads)
From cell volume and abundance we can obtain the total bacterial biomass in µg C/l
following Norland (1993) equation:
µg C L-1 = 0.12 pg µm-3* (vol cell 0.7)* bacterial abundance (L1) / 1000000
b.3)BIOMASS OF PICO AND NANOPLANKTON BY EPIFLUORESCENCE
MICROSCOPY
Dye working solution (DAPI)
-Add 1 ml of filtered seawater (0.2 µm) to a 10 mg DAPI vial.
-Add the 10 mg/ml of DAPI to 19 ml of sterile filtered seawater (0.2 µm). We will
now have a concentration of 0.5 mg/ml de DAPI.
-Filter this solution through 0.2 µm (Swinex and sterile syringes), and put it in a clean
20 ml vial. THIS IS THE WORKING DAPI SOLUTION
-Take 20 criovials and add 1 ml of DAPI working solution to each one. Keep the
criovials frozen (-20 oC) until its use.
How to make slides
a)Bacteria and P. diminuta
-Let a 0.8 µm (25 mm ø, cellulose acetate) filter on the base of the filtration system,
and on top of it put a 0.22 µm (25 mm ø, polycarbonate) filter.
-Adjust the filtration tower and add 5 - 20 ml of the corresponding aliquot.
-With an automatic 5 ml pipette add volumes of 5 ml and filter them. Keep the last 5
ml closing the filtration key.
-Add 50 µl of DAPI working solution (5 µg/ml, final concentration).
-Let the aliquot with DAPI 5-10 minutes and filter.
-Take a slide and note in it: date of sampling, the name of the station, experiment,
depth…
-In each slide, let one or two drops (separately) of low fluorescent oil (Cargille,
Nikon).
10
-With Millipore pincers take each filter from the filtration system, dry it.
-Place the filter (side up) on top of the oil drop. Wait until the filter became
transparent.
-Add another oil drop on top of the filter.
-Put a cover-slide on top of the filter to avoid the formation of air bubbles.
-Keep the slides in special plastic boxes. Note on the box the corresponding name of
the experiment, cruise, and number the box. Keep the slide boxes frozen at -20 oC
-We will dispose of a filtration note book, where we will specify the sampling and
filtration date, the sample code, the filtrate volume, the used filter (0.2 µm), the
preservative volume and the box number.
IMPORTANT: After filtration, keep the leftovers of DAPI working solution in the
freezer for the next day.
b)Nanoflagellates
-Let a 0.8 µm (25 mm ø, cellulose acetate) filter on the base of the filtration system,
and on top of it put a 0.6 µm (25 mm ø, polycarbonate) filter.
-Adjust the filtration tower and add from 10 to 50 ml (depending on the sampling site
or depth) of the corresponding aliquot. It is recommended to filtrate from 20 ml-30 ml
for surface open-sea samples (5-40 m depth), and 30-50 ml for deeper samples. For
coastal samples it is enough to filter 10-20 ml of sample.
-Filter the sample (as for bacteria, using a 5 ml automatic pipette) until the filtration
tower contains 5 ml.
-From here the process to follow is the same as for bacteria.
How to count in the epifluorescence microscope
The available microscopes in the ICM are a NIKON Labophot 2TM and an
OLYMPUS. The objective used for this kind of counts is an immersion oil objective
of100 X. The ocular used has 10 X. The NIKON has an extra magnification of 1.25
X. Thus allowing a final magnification of 1250 X. While for the Olympus is the 1000
X. Inside the ocular there is a calibrated grid which is divided into 100 small squares.
For NIKON the grid has 0.080 mm side, and each small square has 8 µm side. For
Olympus the grid has 0.100 mm side and the size of each small square has 10 µm
side.
Both microscopes are equipped with a Hg lamp of100 W. Each microscope has an
hour counter. The Hg lamp should be changed between 200-400 h of use.
11
Heterotrophic bacteria and nanoflagellates were observed under UV excitation 400
nm and emission 440 nm wavelength. P. diminuta and cells with autofluorescence
(Pico-nano phototrophic) were counted using an optical filter set specific for yellowgreen fluorescence (blue, light, 485nm excitation and 530 nm emission wave-length,
and 505 nm dichroic mirror).
a)Heterotrophic bacteria, Synnechoccocus and P. diminuta
-Unfreeze the slides.
-Turn-on the lamp of the microscope. Wait 5 minutes until the light intensity becomes
stable
-Set the UV filter to observe heterotrophic bacteria stained with DAPI.
-Add a drop of oil (cargylle) on top of the cover-slide.
-Focus.
-Count the needed fields (grid or part of it) to achieve around 200 - 300 cells. In
general with 20 fields is enough.
-Set the blue light filter to observe Synnechoccus (with orange or yellow
autofluorescence) and P. diminuta stained with DTAF (with bright yellow
fluorescence). Both types of cells are very different. Synnechoccocus are spherical
cells and have around 1 µm ø, while P. diminuta are rod shaped cells of 0.8-1 µm
long and 0.3 µm width. Count around 20-30 fields (grids, ~200 cells).
Calculation of cell abundance, cell volume, and biomass
-For this we need to know the microscope factor = Filtration area/ counting area
The filtration area refers to the filter area, hence this will be the area of a circle (pi*
r2). For this we need to know the diameter which will correspond to the tower
filtration diameter. The counting area it refers to the grid area which is a square.
Hence, the counting area will be the square of the grid side
NIKON Factor:
Tower filtration diameter: 21 mm (this will depend of the filtration system used)
Pi: 3.1416
Size grid side (c): 0.080 mm
Factor = Pi * r2/ c2; Factor = 3.1416* 10.5 2 mm2/ 0.0802 mm2 = 54119
OLYMPUS Factor
Tower filtration diameter: 21 mm
Pi: 3.1416
Size grid side (c): 0.100 mm
Factor = Pi * r2/ c2; Factor = 3.1416* 10.52 mm2/ 0.1002 mm2 = 34636
12
-From the average cells per field, the filtered volume and the dilution factor
corresponding to the fixative; cell abundance (ml-1) will be:
(Average cells field-1 * Factor / Filtered volume (ml)) / (dilution factor: Initial sample
volume / Initial sample volume + preservative added) = Cell abundance (cell ml-1)
Example:
Average bacteria field-1: 20
Olympus factor: 34636
Filtered volume: 5 ml
Sample dilution factor: 100 ml/ (100 ml of sample + 10 ml of preservative) = 0.9091
20 *34636/ (5* 0.9091) = 152414 cell ml-1
Epifluorescence counts are needed to compare and test the results obtained by flow
cytometry.
-Cell volume used is the one obtained by flow cytometry (see, Gasol, 1999b).
Bacterial biomass (µg C L-1) is calculated as is shown above (Point b.2).
b)Nanoflagellates
-First, we observe the slides with UV light to be sure that the round particles are cells
with a nucleus. Also, we will try to visualize the flagella when present
-Immediately, change to blue light to discriminate between colorless cells from cells
with plasts and pigments. This makes the possibility to separate heterotrophic from
phototrophic nanoflagellates.
-Counts of each group of nanoflagellates (phototrophic and heterotrophic) are made
separately, enumerating the nanoflagellates through transects (5 - 10 mm). For each
sample it is recommended to count at least 50-100 cells. Hence, we will make the
necessary number of transects (3 transects of 5 or 10 mm are enough).
-At the same time that we perform the counting, we measure the diameter of
nanoflagellates using the micrometric ocular inserted in the ocular. This allows
classifying the nanoflagellates in different size classes (<2 div, 2-5 div, 5-10 div, and
and10-20 div) and for each group of nanoflagellates (phototrophic and heterotrophic).
For Nikon microscope each division of the micrometric ocular is equal to 0.8 µm. For
Olympus each division is equal to 1 µm.
-Using a manual counter (multichannel), for each sample we note the nanoflagellates
group (phototrophic or heterotrophic) and to which size class it belongs.
13
Calculation of nanoflagellate abundance, cell volume and biomass
-Before starting any transect we need to know the nonius position (nonius: rule
attached to the microscope plate). Having done this, we will note the transect's
millimeters
-As for bacteria we need to know the filtration and counting area.
-The filtration area is the same as for bacteria, while the counting area takes into
account the millimetres for each transect. In this case the counting area instead of a
square will be a rectangle.
Factor = Filter area / (side of the grid * mm of the transect)
Factor = Pi* r2 / rectangle area
NIKON factor:
Filtration tower diameter: 21 mm
Pi: 3.1416
Grid side: 0.080 mm
Transect: 5 mm
Factor = Pi * r2/ c2; Factor = 3.1416* 10.52 mm2/ (0.080* 5) mm2 = 866
OLYMPUS
Filtration tower diameter: 21 mm
Pi: 3.1416
Grid side: 0.100 mm
Transect: 5 mm
Factor = Pi * * r2/ c2; Factor = 3.1416* 10.52 mm2/(0.100* 5) mm2 = 692.7
IMPORTANT!!!! The factor will depend on the mm for each transect.
-From average abundance of HNF or PNF per transect, filtered volume and the
dilution factor corresponding to the sample plus preservative (e.g. 100 ml sample/100
ml sample + 10 ml of preservative = 0.9091). Thus cell abundance will be calculated
as:
Average of nanoflagellate transect -1* Factor of the microscope / filtered volume (ml)/
dilution factor = Nanoflagellate abundance (cell ml-1).
14
Example:
Date: 21/5/00
Cruise: Hivern-2000
Experiment: GRZ 1
Microscope: Olympus
1st transect (5 mm)
2-5
5-10
10-20
Total
HNF
47
2
1
50
PNF
58
10
2
70
2nd transect (5 mm)
2-5
5-10
10-20
Total
HNF
47
2
1
50
PNF
58
10
2
70
-The number of cells per transect is calculated summing the number of cells for each
size class.
-Total cells number per ml is calculated averaging the total number of nanoflagellates
of each transect.
50 HNF transecte-1*692.7/ 20 ml / (100 ml/110 ml) = 1905 HNF ml-1
70 PNF transecte-1* 692,7/20 ml/ (100ml/ 110 ml) = 2667.16 PNF ml-1
-Nanoflagellate number per ml of each size class, is calculated averaging the
nanoflagellate number of each size class per transect:
15
Class: 2-5
47 HNF transect-1*692.7/ 20 ml / (100 ml/110 ml) = 1791 HNF ml-1
58 PNF transect-1* 692,7/20 ml/ (100ml/ 110 ml) = 2210 PNF ml-1
Class: 5 -10
2 HNF transect-1*692.7/ 20 ml / (100 ml/110 ml) = 76 HNF ml-1
10 PNF transect-1* 692,7/20 ml/ (100ml/ 110 ml) = 381 PNF ml-1
Class: 10 - 20
1 HNF transect-1*692.7/ 20 ml / (100 ml/110 ml) = 38 HNF ml-1
2 PNF transect-1* 692,7/20 ml/ (100ml/ 110 ml) = 76 PNF ml-1
Total abundance of HNF: 1905 cell ml-1
In order to calculate the biomass we need to know the cell volume and the abundance
for each size class.
-Cell volume is calculated averaging the cell diameter. Thus for the size class between
2-5 µm we take and averaged diameter of 3.5 µm, for the size class of 5-10 µm, the
mean diameter will be 7.5 µm, and for the size class 10-20 µm the mean diameter
taken will be 15 µm. If we are working with the Olympus microscope we know that
each division of the micrometric rule is equivalent to1 µm.
-Once we know the diameter, we will adjust the volume of cells to the volume of a
sphere (4/3* Pi* R3).
-The biomass is calculated as the sum of products of the average volume of each size
class (µm3) multiplied by the abundance of nanoflagellates per litre and for the
conversion carbon factor 0.22 pg µm-3*(1 µg /1000000 pg). This factor of 0.22 pg µm3
is the one described in Børsheim and Bratbak (1987).
Taking the example above:
HNF (2-5) : 1,8*106 cell L-1 *4/3* Pi* 1.753 µm3 * 0.22 pg C µm-3 *1 µg C/ 1000000
pg C = 8.88 µg C L-1
HNF belonging to the size class 2-5 µm represent a 94.2 % of the total abundance and
36% of the total biomass.
16
HNF (5-10): 8.0 * 104 cell L-1 *4/3* Pi* 3.753 µm3 * 0.22 pg. C µm-3 *1 µg C/
1000000 pg C = 3.63 µg C L-1
HNF belonging to the size class 5-10 µm represent a 3.9 % of the total abundance and
14.8 % of the total biomass.
HNF (10-20): 3.8 * 104 cell / L *4/3* Pi* 73 µm3 * 0.22 pg C µm-3 *1 µg C/ 1000000
pg C = 12.01 µg C L-1
HNF belonging to the size class 10 -20 µm represent 1.99 % of the total abundance
and 48.9 % of the total biomass.
Total abundance of HNF: 1905 cell ml-1
Total biomass of HNF: 24.52 µg C L-1
-Total abundance and biomass of PNF will be calculated like HNF.
b.4)GRAZING RATES CALCULATIONS
-Grazing rates and the number of bacterial cells consumed per unit of time and
volume of water is calculated following the mathematical model of Salat and Marrasé
(1994).
g = -1/t* Ln (P. dimt / P. dim o) (Eq. 1)
-1
g: grazing rate (d ); t: incubation time; P. dimt: P. diminuta abundance at a
considered time (8, 12, 24, 36 h) or final (48 h); P. dim o: abundance of P. diminuta at
time zero
a = 1/t * Ln (BHt/BHo) (Eq. 2)
-1
a: net growth rate (d ); t: incubation time; BHt: number of heterotrophic bacteria at a
considered time (8, 12, 24, 36 h) or final (48 h); BHo: number of heterotrophic
bacteria at time zero.
-1 -1
G = g/a * (BHt- BHo)/t = Number of consumed bacteria ml d (Eq. 3)
17
Example:
Number of heterotrophic bacteria and P. diminuta over the incubation period (counts
made by flow cytometry):
DATE
28/2/99
DEPTH
Dupl.
28/2/99
BH (32 h)
BH (44 h)
(cells ml-1)
(cells ml-1)
(cells ml-1)
(cells ml-1)
(cells ml-1)
5.41E+05
2.68E+05
2.26E+05
3.64E+05
9.49E+05
5 m
2
5.41E+05
2.54E+05
2.26E+05
3.78E+05
7.90E+05
5.41E+05
2.61E+05
2.26E+05
3.71E+05
8.70E+05
0.00E+00
7.21E+03
0.00E+00
6.88E+03
7.97E+04
25 m
1
4.17E+05
4.20E+05
5.27E+05
1.07E+06
1.41E+06
25 m
2
6.27E+05
5.32E+05
8.19E+05
1.38E+06
2.49E+06
5.22E+05
4.76E+05
6.73E+05
1.22E+06
1.95E+06
1.05E+05
5.62E+04
1.46E+05
1.52E+05
5.39E+05
P dim (0 h)
P dim ( 9 h )
P dim(20h)
P dim(32h)
P dim(44h)
(cells ml-1)
(cells ml-1)
(cells ml-1)
(cells ml-1)
(cells ml-1)
DEPTH
Dupl.
5 m
1
1.91E+05
1.72E+05
1.62E+05
1.36E+05
1.18E+05
5 m
2
1.91E+05
1.88E+05
1.62E+05
1.76E+05
1.59E+05
1.91E+05
1.80E+05
1.62E+05
1.56E+05
1.39E+05
0.00E+00
8.10E+03
0.00E+00
1.96E+04
2.05E+04
AVG
28/2/99
BH (20 h)
1
AVG
DATE
BH (9 h)
5 m
AVG
28/2/99
BH (0 h)
25 m
1
1.73E+05
1.73E+05
1.50E+05
1.44E+05
1.27E+05
25 m
2
1.69E+05
1.71E+05
1.50E+05
1.50E+05
1.31E+05
1.71E+05
1.72E+05
1.50E+05
1.47E+05
1.29E+05
2.09E+03
9.00E+02
0.00
3.08E+03
1.72E+03
AVG
18
Net growth rates (a, d-1) and grazing rates (g, d-1) over the incubation period. Results
come from equations 1 and 2:
DATE
28/2/99
DEPTH
Duplicates
28/2/99
a(0-32)
a(0-44)
( d - 1)
( d - 1)
( d - 1)
( d - 1)
1
-1.87E+00
-1.05E+00
-2.97E-01
3.07E-01
5 m
2
-2.01E+00
-1.05E+00
-2.69E-01
2.07E-01
-1.94E+00
-1.05E+00
-2.83E-01
2.57E-01
7.14E-02
0.00E+00
1.42E-02
5.01E-02
25 m
1
1.91E-02
2.81E-01
7.07E-01
6.66E-01
25 m
2
-4.37E-01
3.21E-01
5.92E-01
7.54E-01
-2.09E-01
3.01E-01
6.49E-01
7.10E-01
2.28E-01
1.98E-02
5.75E-02
4.39E-02
AVERAGE
DATE
a(0-20)
5 m
AVERAGE
28/2/99
a(0-9)
DEPTH
Duplicates
g(0-9)
g(0-20)
g(0-32)
g(0-44)
( d - 1)
( d - 1)
( d - 1)
( d - 1)
5 m
1
2.79E-01
1.98E-01
2.55E-01
2.63E-01
5 m
2
4.21E-02
1.98E-01
6.13E-02
1.00E-01
1.60E-01
1.98E-01
1.58E-01
1.82E-01
1.18E-01
0.00E+00
9.67E-02
8.15E-02
AVERAGE
28/2/99
25 m
1
0.00E+00
1.71E-01
1.38E-01
1.69E-01
25 m
2
0.00E+00
1.43E-01
8.94E-02
1.39E-01
0.00E+00
1.57E-01
1.14E-01
1.54E-01
0.00E+00
1.40E-02
2.41E-02
1.49E-02
AVERAGE
19
Consumed cells per ml, day and each considered time (Eq. 3):
DATE
28/2/99
DEPTH
Duplicates
G(0-20)
G(0-32)
G(0-44)
Cell ml- 1 d -1
Cell ml- 1 d -1
Cell ml- 1 d -1
Cell ml- 1 d -1
5 m
1
1.09E+05
7.13E+04
1.14E+05
1.91E+05
5 m
2
1.60E+04
7.13E+04
2.79E+04
6.58E+04
6.23E+04
7.13E+04
7.08E+04
1.28E+05
4.63E+04
0.00E+00
4.30E+04
6.25E+04
AVERAGE
28/2/99
G(0-9)
25 m
1
0.00E+00
8.04E+04
9.54E+04
1.37E+05
25 m
2
0.00E+00
1.03E+05
8.54E+04
1.88E+05
0.00E+00
9.17E+04
9.04E+04
1.63E+05
0.00E+00
1.12E+04
4.99E+03
2.51E+04
AVERAGE
As a final value of grazing on bacteria per ml and day, we will take the one that is
constant during different incubation times. Thus the value for 5 m will be 7.1 x 104
cells ml-1 and d-1 and for 25 m will be 9.1 x 104 ml-1 d-1.
Calculation of the percentage of bacterial standing stock and bacterial production
consumed (d-1)
Bacterial consumption can be given as number of bacteria grazed (cell ml-1 d-1), as
well in percentage of the bacterial standing stock as on bacterial production.
a)On bacterial standing stock
The percentage of bacterial consumed on the standing stock (d-1), can be calculated on
bacterial biomass whether with respect to initial bacterial abundance or to initial
bacterial carbon.
-The percentage of bacterial abundance consumed per day will be calculated as:
G (Cell ml-1 d-1)* 100/ BHo (cell ml-1) = % of consumed bacteria per day.
20
-To obtain the bacterial carbon percentage consumed per day, we firstly need to
calculate the bacteria consumed per day in terms of carbon (µg C l-1). For this we use
the cell volume obtained by flow cytometry and as we did before by means of the
Norland (1993) equation we will obtain the bacterial biomass (µg C L-1) and the
bacterial carbon consumed at different times (µg C L-1 d-1).
-The percentage of bacterial carbon consumed per day will be calculated as:
G (µg C L-1 d-1)* 100 / BHo (µg C L-1) = % of bacterial carbon consumed per day
b)On bacterial production
The percentage of bacteria consumed on bacterial production (d-1) can be calculated
whether with respect to the carbon bacterial production or to cells bacterial
production.
-The percentage of bacterial carbon consumed to the bacterial carbon produced can be
calculated:
i)from the bacterial carbon production within the incubation bottles as:
Gt +(Bct-Bco)/t. Then the percentage will be:
% Bact. production consumed (d-1) = G (µg C L-1 d-1)* 100 / [(G +( Bct-Bco)/ t].
ii)From the bacterial carbon production obtained by tritiated leucine incorporation
(PBleu, µg C l-1 d-1). Then the percentage will be:
% Bact. production consumed (d-1) = Gt (µg C L-1 d-1)* 100 / (PBleu, µg C L-1 d-1)
-The percentage of bacterial cells consumed to the bacterial cells produced can be
calculated as before but in both cases, grazing on bacteria (G), bacterial biomass and
production has to be expressed in cells. For bacterial production, when leucine
incorporation is used we have to convert bacterial carbon in cells (See Gasol et al.
1999a).
Notice: Bacterial production determined in the incubation bottles is usually higher
than bacterial production measured by 3H leucine. This is due to the incubation time
which for tritiate leucine is around three hours, while for bacterial production obtained
within the incubation bottles (function of grazing and net bacterial growth) require
longer incubation time. If both types of bacterial production are correlated we can
extrapolate the percentage of the bacterial production grazed obtained in the
incubation bottles to the bacterial production obtained by 3H leucine.
21
Specific grazing (BH consumed by HNF and per hour)
-If we assume that heterotrophic nanoflagellates are the main bacterial consumers we
can estimate the number of bacteria consumed per flagellate and per hour. Thus, from
the consumed bacteria (G, ml-1 d-1) divided by the abundance of HNF per ml and 24 h
we will obtain:
BH HNF-1 h-1 = G (BH ml-1 d-1)/ HNF ml-1/ 24 h
Phototrophic picoplankton
Although we have not mentioned grazing on phototrophic picoplankton
(Synnechoccocus, Prochlorophytes and Picoeucaryotes), it is very interesting to have
this data in order to interpret the grazing data. When Synnechococcus abundance is
important, there are protocols to determine grazing rates on these microorganisms. For
that we will use fluorescent labeled cells called FLA. The methodology used is similar
to that of bacterial grazing. (We will describe the correspondent protocol soon so it
can be used for everybody.)
b.5)CILIATES ABUNDANCE AND BIOMASS USING AN INVERTED
MICROSCOPE
-From the lugol acidic fixed sample, 100 ml is sedimentated for 48 h in a
sedimentation chamber under the hood to avoid noxious gases.
-The settling material is collected in the base of the chamber, removing the
supernatant.
-Counts of ciliates are made in an inverted microscope (Zeiss Axiovert 35) at 200X or
400X.
-Turn on the lamp.
-Use a contrast phase filter.
-Use the adequate objective (20-40X) and ocular of 10X. Hence we will have a
magnification of 200-400X.
-Place the chamber in the plate of the microscope, focus the sample, and as for
nanoflagellate counts make several transects.
-In a worksheet we will note date, sample source, magnification used (200X or 400X)
and the position where we start the transect (look at the position of the nonius).
-Make a minimum of 8 transects of 10-15 mm.
22
Abundance, cell volume and biomass of ciliates
-For each transect made we will write down the ciliate numbers of each group that we
are capable of identifying, and with the micrometric rule inserted in the ocular we will
measure width and lengths of each ciliate (these not include the cilia). Alternatively:
we can count all ciliates first, and once finished counts, we can measure a determined
number of cells (minimum 30 cells).
Lengths and widths are noted beside the numbers in brackets.
EXAMPLE
DATE
3/5/00
Microscope
Zeiss
Sample
Blanes
Magnification
400
ø sed chamber
25 mm
Ciliates
Halteria
Strombidium
Transect
Transect
Transect
15 mm
15 mm
15 mm
4 (5)
1(5)
5 (5,3)
6 (8,5)
Laboea
Tontonia
Strobilidium
5 (8)
Tintinnids
2 (12,3)
Unknown
SUM
9
6
8
AVERAGE Cell Transect-1: (9+6+8)/3 = 7.7 cell Transect-1
-From the average cell Transect-1 we can calculate the number of ciliates per litre. For
this we need to know the microscope factor, the sedimented volume and the mm of
each transect.
Factor for 400X
Sedimentation chamber area/ counting area:
-The sedimentation chamber has 25 mm hence its surface is:
23
Pi *r2 = 3.1416 * 12.5 mm2
-Counting area is the field surface: 0.481 mm2
-For a transect of 1 mm we will have a Factor of 1020
FACTOR = 3.1416 * 12.52 mm2/ 0.481 * 1 mm2 = 1020
FACTOR
1020
Transect
15 mm
Volume sed.
100 ml
Total Ciliate/L
5213
Following with the above example:
Cil L-1 = 7.7 Cil (Transect-1) * 1020/15 mm (Transect-1) *1000 ml/1L*1/100 ml =
5213 Cil L-1
To calculate the ciliate abundance for each group, we will use the same formula and
the average ciliate of each group transect-1.
Ciliate
Avg./trans
Cells/Litre
Halteria
1.67
1133
Strombidium
3.67
2493
Laboea
0
0
Tontonia
0
0
Strobilidium
1.67
1133
Tinitinnids
0.67
453
Unknown
0
0
SUM
5213.333
24
Cell volume: We adjust the volume of each cell to the volume of the nearest
geometric figure.
VOLUME CELL OF Geom.fig.
Halteria
Sphere
Strombidium
Ellipse
Laboea
Ellipse
Tontonia
Ellipse
Strobilidium
Sphere Ellipse
Tintinnids
Sphere Ellipse
Conical
At 400 X each division of the ocular micrometric measure 2.42 µm. (All these
parameters, like factors, division values can be found in the microscope Zeiss room).
Ciliate Types
Cells/Litre
Average
Bio volume
Biomass
vol cell
µm3
µm3 L- 1
µg C L- 1
Halteria
1133
927
1.05E+06
2.10E-01
Strombidium
2493
961
2.40E+06
4.79E-01
1133
3799
4.31E+06
8.61E-01
Tintinnids
453
801
3.63E+05
2.11E-02
Unknown
0
8.12E+06
1.57E+00
Laboea
0
Tontonia
0
Strobilidium
SUM
5213
Example to calculate cell volume:
Halteria (Sphere)= 4/3*3.1416* 2.53*2.423 = 927 µm3 cil-1
-If we find different sizes for the same type of ciliate like for Strombidium we will
average their volumes and we will obtain the mean cell volume. Thus, if there are 5
cells that have 5 division length and 3 divisions width (5,3), and 6 cells that have 8
25
divisions length and 5 width, after applying the corresponding volume formula (in this
case an ellipse), we will average these volumes.
Biomass calculation:
-The biovolume for a determined group of ciliates (µm3 L-1) is obtained multiplying
the abundance of each group by the mean volume cell. Biovolume for the total ciliate
community will be calculated summing all partial biovolumes of different ciliate
groups.
-Partial or total biovolume will be converted to biomes multiplying by a conversion
carbon factor from the literature of 0.2 pg C µm-3 valid for all groups (Putt and
Stoecker, 1989) except for tintinnids. For this group the used factor is 0.053 pg C µm-3
(Verity and Langdon, 1984)
26
ANNEX. NECESSARY MATERIALS FOR:
a) SAMPLING
Sample collection
-Plastic carboys of 5 L
-Nytex net of 50 µm
-Plastic bottles of 125 ml
-Graduated cylinders of 50, 100 ml. and 1 Litre
-Pipettes of different volumes (50 µl to 5 ml)
-Plastic funnel
-Silicone sampling tubs
Incubations
-Incubator bath or chambers (for different temperatures)
-Net of different width to simulate light conditions
-Hose and connections
-Adequate illumination system
-PAR system to measure the incident light
-Thermometre
-Plastic bottles of 1.5 l
b) BACTERIAL PRODUCTION
(See Gasol 1999a)
c) FLOW CYTOMETRY
(See Gasol 1999b)
d) EPIFLUORESCENCE
Dyes
-DAPI (4,6- diamidino-2-phenylindole, 10 mg Sigma)
-FITC Fluorescein-Isothiocyanate (50 mg, Sigma F-7250)
-DTAF 5-([4-6 dichlorotriazin-2-yl] amino) fluorescein (100 mg, Sigma D-0531)
27
Preservatives
-Glutaraldehyde (25 %, Merck). Make a solution at 10%
Preparation: Filter 600 ml of filtered seawater through 0.2 µm
Add 400 ml of glutaraldehyde (25%)
Filter again through 0.2 µm
Keep the solution in the refrigerator at 4 oC
-Dispenser of Glutaraldehyde solution (10%)
Filtration and samples observation
-Plastic bottles of 125 ml
-Graduated cylinder of 50 ml
-Pipette of 5 ml
-Pipette of 10-100 µl
-Millipore pins
-Black polycarbonate filters of 0.2 µm (25 mm of diameter, Millipore)
-Black polycarbonate filters of 0.6 µm (25 mm of diameter, Nuclepore)
-Acetate cellulose filters of 0.8 µm (25 mm of diameter, Millipore)
-Acetate cellulose filters of 0.2 and 0.8 µm (47 mm de diameter, Millipore)
-Filtration flask of 1L or 2L.
-Filtration set
-Pressure Pump
-Wastewater container
-Silicone tubs
-Standard slides (76 x 26 mm)
-Cover-slides of 24 x 24 mm
-Immersion oil of low fluorescence (DF MXA20351, Nikon)
-Plastic slide boxes
-Freezer of -20 oC (at least)
-Epifluorescence microscope
-Notes book
e) CILIATE COUNTS
Collection and sedimentation of samples
-Plastic bottles of1.5 L
28
-Graduated cylinder of 1L
-Sedimentation chambers (100 ml)
-Notes book
Preservation
-Dispenser or pipette
-Acetic lugol:
H20 distilled 1 L
Ac. glacial acetic
Potassium iodide
Iodine
100 ml
100 g
50 g
Filter the solution with filter paper
Mark on the bottle the preparation date
Keep refrigerated
Sample observation
-Inverted microscope. Transmission light
RELEVANT BIBLIOGRAPHY
Børsheim, K. Y.; Bratbak, G. 1987. Cell volume to cell carbon conversion factors for
a bacterivorous Monas sp. enriched from seawater. Mar. Ecol. Prog. Ser. 36: 171-175.
Gasol, J.M. 1999a. How to measure bacterial activity and production with the uptake
of radiolabeled leucine.
ftp://ftp.icm.csic.es/pub/gasol/Manuals/ProdBact/Leucine.htm (Web page address).
References therein.
Gasol, J.M. 1999b. How to count picoalgae and bacteria with the FACScalibur flow
cytometer.
ftp://ftp.icm.csic.es/pub/gasol/Manuals/FACS/Citometry.html (Web page address).
References therein.
Maniatis, T.; Fritsch, E.F.; Sambrook, J. 1982. Molecular cloning: a laboratory
manual. Cold Spring Harbor Laboratory. Press. NY.
29
Norland, S. 1993. The relationship between biomass and volume of bacteria. In:
Kemp, P.F.; Sherr, B.F.; Sherr E.B.; Cole, J.J. (eds.). Handbook of methods in aquatic
microbial ecology, p. 303-307. Lewis Publishers. Boca Raton.
Porter, K.G.; Feig, Y. S. 1980. The use of DAPI for identifying and counting the
aquatic microflora. Limnol. Oceanog. 25: 943-948.
Putt, M.; Stoecker, D.K. 1989. An experimentally determined carbon: volume ratio
for marine oligotrichous ciliates from estuarine and coastal waters. Limnol. Oceanogr.
34: 1097-1104.
Salat, J.; Marrasé, C. 1994. Exponential and linear estimations of grazing on bacteria:
effects on changes in the proportion of marked cells. Mar. Ecol. Prog. Ser. 104:205209.
Vaqué, D.; Pace, M.l.; Findlay, S.E.G.; Lints, D. 1992. Fate of bacterial production in
a heterotrophic ecosystem: grazing by protist and metazoans in the Hudson estuary.
Mar. Ecol. Prog. Ser. 89:155-163.
Vaqué, D.; Gasol, J. M.; Marrasé, C. 1994. Grazing rates on bacteria: the significance
of methodology and ecological factors. Mar. Ecol. Prog. Ser. 109: 263-274.
Vaqué, D.; Blough, H. A.; Duarte, C. M. 1997. Dynamics of ciliate abundance,
biomass and community composition in an oligotrophic coastal environment (NW
Mediterranean). Aquat. Microb. Ecol. 12: 71-83.
Vazquez-Dominguez, E. P.; Gasol, J.M.; Peters, F.; Vaqué, D. 1999. Measuring the
grazing losses of picoplankton: Methodological improvements to the use of
fluorescently labeled tracers combined to flow cytometry. Aq. Microbial Ecol. 20:
110-128. References therein.
Verity, P.G.; Langdon, C. 1984. Relationships between lorica volume, carbon,
nitrogen and ATP content of tintinnids in Narragansett Bay. J. Plankton Res. 6: 859868.
ACKNOWLEDGMENTS. I am grateful to Clara Cardelús for correcting the text
30