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Groben & Medlin In situ hybridisation of phytoplankton

Tools and Approaches in Eukaryotic Microbial Ecology


in press
15.4. IN SITU HYBR!"AT#$ #% &HYT#&'A$(T#$ )"$*
%')#RE"+E$T'Y, 'ABE''E! rR$A &R#BE"
Ren- *roben . 'inda (. Medlin
15.4.1. ntroduction
Molecular biological techniques have greatly enhanced our ability to analyse all types
of organisms, including the microalgae. This represents a major step forward in
marine oceanography, as many microalgae are small in size, lack distinct
morphological markers, and are unculturable, which makes it difficult to estimate
their biodiversity. The lack of knowledge of their breeding systems also makes
genetic or demographic studies difficult, too, and in addition, long term seasonal
studies in aquatic environments are problematic for logistic reasons. This has hindered
our understanding of microalgal diversity and their population structure. Despite this,
physiologicalbiochemical measurements have been used to infer the e!istence of
significant genetic diversity within and between microalgal populations (1-3). "ith
these data researchers have speculated on hidden biodiversity and temporal and
spatial structuring of genetic diversity or gene flow. #ow molecular techniques can
present a quantitative framework through which the diversity, structure and evolution
of microalgal populations can be analysed, predictive models of the dynamics of
aquatic ecosystems formulated, and the idea of functional groups in the plankton
proven.
#evertheless, molecular analysis of microalgal population structure is behind other
groups and has been usually inferred from physiological data determined from
relatively few clones. This unfortunately is a very naive approach because nearly
every physiological measurement has shown that no single clone of any microalgal
species can be considered truly representative of that species (4).
The interaction of a species with environmental parameters is influenced by the
genetic diversity at the population level of a species. $patial and temporal partitioning
%
Groben & Medlin In situ hybridisation of phytoplankton
of genetic diversity will occur as these interactions structure the ecosystem. $uch
structuring has seldom been measured in the microalgal community and studies of
genetic diversity are virtually non&e!istent in pelagic ecosystems. 'll evidence of
geographically isolated populations would be erased if we continue to assume that
microalgae with high dispersal capacities are genetically homogeneous over their
entire range. $upport for this assumption has come mainly from phenotypic
comparisons based initially on net phytoplankton biogeographic studies and later on
isozyme studies. (t is clear that the same morphotypespecies may be endemic or
cosmopolitan (5) but it is more likely that cosmopolitan species will e!hibit regional
differentiation when e!amined with molecular techniques (6).
)arp et al. *7+ provide an e!cellent introduction into the variety of molecular
techniques available for use in studying biodiversity at all ta!onomic levels, whereas
other useful reviews deal with the biodiversity in the marine environment *8+ and in
the marine phytoplankton *9+. "e present below a brief review of the progress in
analyzing microalgal populations beginning first at the population level and building
to higher ta!onomic levels with the use of r,#' probes. ' detail protocol of
-luorescent In Situ .ybridisation *-($.+ methods applicable for most algal cell types
so far tested is presented at the end of the chapter.
15.4./. !$A 0ingerprinting
/ur current limited knowledge of microalgal genetic diversity is a direct consequence
of the difficulties of finding polymorphic markers for ecological genetic studies.
(sozymes, the molecular markers used in early studies, evolve so slowly that closely
related populations appear identical. The early viewpoints suggesting the absence of
genetic diversity in microalgae have undoubtedly been propagated from these studies.
The use of high resolution D#' fingerprinting techniques sensu lato circumvents
these problems and has thus opened areas previously considered unreachable for the
microalgae.
D#' fingerprinting is a generic term for different molecular techniques, which have
in common that they produce multi&locus banding patterns and can be used to analyse
populations down to individuals. The most commononly used techniques are ,'0Ds
*,andom 'mplified 0olymorphic D#'s+, '-10s *'mplified -ragment 1ength
0olymorphisms+,2#T,s *2ariable #umber of Tandem ,epeats3 minisatellites+ and
$T,s *$imple Tandem ,epeats3 microsatellites+ (7). "hile these methods are used for
4
Groben & Medlin In situ hybridisation of phytoplankton
population studies in many higher eukaryotic organisms, their employment for
analyzing microalgae is still limited thus far. The few e!amples of studies of
biodiversity and population genetics in phytoplankton include those of the
prymnesiophyte Emiliania huxleyi (10) and the dinoflagellate Symbioinium (11),
both analysed by ,'0Ds, as well as the dinoflagellate species !lexan"ium
tama"ense (1#), which was investigated using '-10s. The latter e!ample showed that
these types of fingerprints can provide ironically too much variation in the case of
population studies because of the high variability at each locus and the large number
of loci. 5anding patterns can quickly become so comple! that they cannot be
analyzed in terms of allele frequencies *the data of population genetic measures+.
/bviously, more effort is necessary to establish these techniques and more knowledge
about the genetic structure of most phytoplankton species must be gained, before
D#' fingerprinting methods can be routinely used to investigate phytoplankton
biodiversity and population structures. Thus far, D#' fingerprinting with 2#T,s
hasn6t been used in microalgal analysis to our knowledge, but single locus
microsatellite markers were applied in analyzing the diatom $itylum b"i%ht&ellii *13+
and E. huxleyi *14+.
15.4.1. "e2uence data
$equence data for both coding and non&coding regions of the genome can be used to
reconstruct the evolutionary history of organisms and to e!amine relationships at all
ta!onomic levels. The ribosomal ,#' genes are usually used for phylogenetic
analyses, although many genes are potentially available. The r,#' genes have special
attributes that make them ideally suited as molecular markers *15+. They are of a
relatively large size, contain both variable and highly conserved regions to address
both close and distant evolutionary relationships, respectively, and are of an
universally conserved function with no evidence to suggest that they are laterally
transferred *15+.
#on&coding regions separating genes are termed spacer regions. (n some
operons, such as in the ribosomal operon, they function in the final processing of the
mature r,#' molecular but in most other genes their function is not well understood.
They can evolve at a faster rate because they are not subjected to the same
evolutionary constraints as coding regions. To resolve closely related species or
population level genetic structure, these faster&evolving non coding regions are best
7
Groben & Medlin In situ hybridisation of phytoplankton
used but even they can be conserved at the genus level or higher in some algae
*reviewed in 6+.
'nalysis can be performed on the sequences obtained form mi!ed natural
samplescommunities. "hole D#' e!traction of a water sample, followed by cloning
of 08,&amplified rD#' sequences and screening of random clones from this library
can provide insights into the genetic diversity, which are unobtainable using more
traditional means of community analyses. This is especially true for unculturable
groups. (n every such study novel groups have been found such that the biodiversity
of the picoeukaryotic fraction in oceanic samples is considerably underestimated as
has found to be the case for the prokaryotic fraction.
5iases in the phylogenetic results obtained will vary with choice of gene used,
the geological age of the ta!a investigated, the rate of evolution in the gene of choice,
the number of nucleotides used in the analysis, the number of outgroups, the
evolutionary model and the ta!a selected for analyses. selected The Model test
program *http9bioag.byu.eduzoologycrandall:labmodeltesr.htm+ tests ;< different
types of models of evolution to determine the model that best fits the data so that this
model and all of its parameters can be imported into 0'=0 for analysis. 5ut
nevertheless phylogenies have been generated for most microalgal groups and they
are under frequent refinement. (n the last %> years, three new microalgal classes have
been either defined or recognised from sequence analysis *16-18+.
15.4.4. #ligonucleotide probes 0or the detection o0 phytoplankton
The fast and secure identification of phytoplankton, especially of to!ic species, is
important from an ecological and economical point of view, but the aforementioned
problems for most pico& and nanoplankton species makes this difficult. (dentification
usually necessitates other time and cost intensive techniques, such as electron
microscopy, pigment analysis with high&performance liquid chromatography *.018+
or sequencing of conserved genes before a definitive identification can be made of
particularly difficult ta!a. 0hytoplankton species identification by whole&cell
hybridisation with specific fluorochrome&labelled probes followed by fluorescence
microscopy or flow cytometry offers a faster alternative for species identification.
5ased upon conserved and variable regions of the ,#' of the ribosomal small and
large subunit *$$=, 1$= r,#'+, signature sequences of varying specificity can be
found, which have been used to develop probes for the identification of phytoplankton
?
Groben & Medlin In situ hybridisation of phytoplankton
at various ta!onomic levels from classes down to species or strains. The vast amount
of rapidly accumulating sequence data for all kinds of organisms makes it possible to
develop these probes for a broad spectrum of ta!a. 'lthough these techniques have
been largely used for 5acteria *i.e., 19' #0+ *see also 8hapter @+ there is already a
growing number of probes for eukaryotic pico& and nanophytoplankton. To date, they
have been developed for classes including 8hlorophyceae *#1' ##+,
0rymnesiophyceae *##' #3+, 0elagophyceae *##+, Dinophyceae *4A+ and
5olidophyceae *#4+, ta!onomic clades like those for to!ic and non&to!ic
(h"yso)h"omulina*"ymnesium species *#5+ and for ecologically& and economically&
important species like (h"yso)h"omulina +olyle+is *#5+, !lexan"ium tama"ense *#6+,
!. osten,elii *#7+, *haeo)ystis %lobosa *#3+, Emiliania huxleyi *#8+ and various
*seuonit-s)hia species *#6+. 's mentioned before, even more probes are available
for numerous procaryotic groups and strains, including many marine and limnic
bacteria. 'n overview of the analysis of these groups by r,#' probes is given by
'mann et al. *#9+. $uccessful application of molecular probes to field samples have
already demonstrated their use in characterization of phytoplankton abundance, e.g.,
for *seuo-nit-s)hia species in coastal waters from 1ouisiana, =$' *30+,
5olidophyceae in the Mediterranean $ea and the 0acific /cean *#4+ or groups of
0rymnesiophytes also in the 0acific /cean *#8+.
Ex+e"imental (onsie"ations
The broad diversity we face in the phytoplankton makes it difficult to develop an in
situ protocol capable of analysing all kinds of algal cells. -or e!ample, different types
of cell walls and membranes may require different conditions for probe penetration.
'lso, cell autofluorescence, especially from chlorophyll, can become a problem when
it is very strong and therefore masks the probe signal. Taken these problems into
account, we adapted an e!isting protocol *31' 3#+ for in situ hybridisation with
specific, fluorescent&labelled probes to suit a broader range of phytoplankton species.
-or the fi!ation and hybridisation of phytoplankton using fluorescent&labelled
probes, various protocols are in use *i.e., ##' 31+, that are mainly derived from those
developed for bacteria and often use paraformaldehyde *0-'+ as the fi!ative *#0+.
8omparing different protocols we found that one published by $cholin et al. *31' 3#+
gave the best results with most species tested. This method eliminates
paraformaldehyde and replaces it with a saline ethanol fi!ative. #evertheless, this
;
Groben & Medlin In situ hybridisation of phytoplankton
method was originally developed for probes for two genera only and including at least
two mismatches between target and non&target sequence. "e found the conditions in
our own e!periments not to be stringent enough for a broader range of species and
probes. Thus we modified the hybridisation conditions to increase stringency by
addition of formamide to the hybridisation buffer and reducing the salt concentration
in the last washing step. -ormamide concentrations must be established empirically
for every probe and normally range between > and ;>B.
During the testing of different fi!ationhybridisation protocols it became clear that
one of the most important components was the type of detergent used. $odium
dodecylsulfate *$D$+, for e!ample, which is often used in hybridisation buffers *i.e.,
##+ destroys the more fragile cells like unarmoured dinoflagellates, whereas (CD0'1&
8'<7> *or the chemically identical #/#(DDT&0?>+ maintains cell stability whilst
enabling efficient probe penetration into the cell. The use of the latter detergent in the
hybridisation buffer, enables the investigation of some of the most delicate
dinoflagellates, such as .ymnoinium mi/imotoi *-ig. % a E b+.
(n addition to the fi!ation, the saline ethanol in the $cholin protocol *31' 3#+ e!tracts
the chlorophyll from the cells and bleaches them, thus enabling good visualisation of
probe signals. This is an advantage for in situ hybridisation e!periments with
phytoplankton in which autofluorescence can be problematic. #evertheless,
sometimes the autofluorescence of some species is so strong and persistent that a sole
ethanol treatment, even for an prolonged time, is not sufficient for probe detection
using fluorescence microscopy *-ig. % c+. (n these cases, an additional treatment with
;>B dimethylformamide *DM-+ can help, because it bleaches the chlorophyll from
the cells far better than ethanol alone *-ig. % d+. 's DM- is to!ic, this procedure
should be added to the protocol only if autofluorescence of cells is e!pected to be a
problem.
=sing this modified protocol it is possible to analyse a very broad range of laboratory
cultures and field samples with in situ hybridisation using probes ranging from single
to multiple base mismatches. This provides researchers with a powerful tool for
investigating the occurence and biodiversity of phytoplankton.
<
Groben & Medlin In situ hybridisation of phytoplankton
A+($#3'E!*EME$T"
The authors want to thank C. )irst *=niversity of 5remen+ for his helpful ideas about
the DM- treatment. This work was funded in part by D= 0(8/D(2 D2)7&8T&%FFF&
>>>4%.
RE%ERE$+E"
1. "aterbury, G.5., "atson, $."., Cuillard, ,.,.1. and 5rand, 1.D. *%FAF+
"idespread occurrence of a unicellular, marine planktonic cyanobacterium.
0atu"e 4AA, 4F7&4F?.
#. 5rand, 1.D. *%F@F+ ,eview of genetic variation in marine phytoplankton species
and the ecological implications. 1iol. 2)eano%". <, 7FA&?>F.
3. 0artensky, -., .oepffner, #., 1i, ".)."., =lloa, /. and 2aulot, D. *%FF7+
0hotoacclimation of *"o)hlo"o)o))us s+. *0rochlorophyta+ strains isolated from
the #orth 'tlantic and the Mediterranean $ea. *lant *hysiol. %>%, 4@;&4F<.
4. "ood, '.M. and 1eatham, T. *%FF4+ The species concept in phytoplankton
ecology. 3. *hy)ol 4@, A47&A4F.
5. )ristiansen, G. *4>>%+ 5iogeography of silica&scaled chrysophytes. 0o4a
5e&i%ia 1eih. %74' 47&7F.
6. Medlin, 1.)., 1ange, M., Ddvardsen, 5. and 1arsen, '. *4>>>+ 8osmopolitan
flagellates and their genetic links, in 6he 7la%ellate !l%ae *Creen, G.8. and
1eadbeater, 5.$.8., eds.+ -rancis and Taylor, 1ondon, pp. 4@@&7>@.
7. )arp, '., (saac, 0.C. and (ngram, D.$. *%FF@+ 8ole)ula" tools ,o" s)"eenin%
bioi4e"sity. 8hapman E .all, 1ondon, =.).
8. /rmond, ,.-., Cage, G.D. and 'ngel, M.2. *%FF@+ 8a"ine 1ioi4e"sity9 *atte"ns
an *"o)esses. 8ambridge =niversity 0ress, 8ambridge.
9. Medlin, 1.)., 1ange, M. and #oethig, D.2. *4>>>+ Cenetic diversity of marine
phytoplankton9 a review and a look to 'ntarctic phytoplankton. !nta")ti). S)i' %4,
74;&77%.
10. 5arker, C.1.'., Creen, G.8., .ayes, 0.). and Medlin, 1.). *%FF?+ 0reliminary
results using the ,'0D analysis to screen bloom populations of Emiliania huxleyi
*.aptophyta+. Sa"sia AF, 7>%&7><.
A
Groben & Medlin In situ hybridisation of phytoplankton
11. 5aillie, 5.)., 5elda&5aille, 8.'., $ilvestre, 2., $ison, M., Comez, '.2. and
Monje, 2. *4>>>+ Cenetic variation in Symbioinium isolates from giant clams
based on random&amplified&polymorphic D#' *,'0D+ patterns. 8a". 1iol. %7<,
@4F&@7<.
1#. Gohn, =., Medlin, 1.). and Croben, ,. *4>>7+ Development of 'mplified
-ragment 1ength 0olymorphisms *'-10s+ to analyse clades within the
!lexan"ium tama"ense species comple!. Eu". 3. *hy)ol., under revision
13. ,ynearson, T.'. and 'rmbrust, D.2. *4>>>+ D#' fingerprinting reveals e!tensive
genetic diversity in a field population of the centric diatom $itylum b"i%ht&ellii.
:imnol. 2)eano%".?;, %74F&%7?>.
14. (glesias&,odriguez, M.D., Carcia $Hez, '., Croben, ,., Ddwards, ).G., 5atley, G.,
Medlin, 1.). and .ayes, 0.). *4>>4+ 0olymorphic microsatellite loci in global
populations of the marine coccolithophorid Emiliania huxleyi. 8ol. E)ol. 0otes 2,
495-497.
15. "oese, 8.,. *%F@A+ 5acterial evolution. 8i)"obiol. ;e4. ;%, 44%&4A%.
16. 'nderson, ,.'., $aunders, C."., 0askind, M.0. and $e!ton, G.0. *%FF7+ The
ultrastructure and %@$ r,#' gene sequence for *ela%omonas )al)eolata gen. E
sp. nov., and the description of a new algal class, the 0elagophyceae classis nov. 3.
*hy)ol. 4F, A>%&A%;.
17. Cuillou, 1., 8hretiennot&Dinet, M.&G., Medlin, 1.)., 8laustre, .., Coeer, $.1.&D.
and 2aulot, D.' *%FFF+ 1oliomonas9 a new genus with two species belonging to a
new algal class, the 5olidophyceae *.eterokonta+ 3. *hy)ol. 7;, 7<@&7@%.
18. )awachi, M., (nouye, (., .onda, D., /I)elly, 8.G., 5ailey, G.8., 5idigare, ,.,. and
'ndersen, ,.'., *4>>4+. The 0inguiophyceae classis nova, a new class of
photosynthetis strameophiles whose member produce large amounts of omega&7
fatty acid. *hy)ol. ;es. ;>, 7%&?A.
19. $tahl, D.'. and 'mann, ,. *%FF%+ Development and application of nucleic acid
probes, in 0u)lei) !)is 6e)hni<ues in 1a)te"ial Systemati)s *$tackebrandt, D.
and Coodfellow, M., eds.+ Gohn "iley E $ons 1td., 8hichester, =.)., pp. 4>;&
4?@.
#0. 'mann, ,. (. *%FF;+ In situ identification of micro&organisms by whole cell
hybridization with r,#'&targeted nucleic acid probes, in 8ole)ula" 8i)"obial
@
Groben & Medlin In situ hybridisation of phytoplankton
E)olo%y 8anual 3.3.6. *'kkermans, '.D.1., van Dlsas, G.D. and de 5ruijn, -.G.,
eds.+ )luwer 'cademic 0ublishers, Dordrecht, #1, pp. %&%;.
#1. $imon, #., 1e5ot, #., Marie, D., 0artensky, -. and 2aulot, D. *%FF;+. -luorescent
in situ hybridization with r,#'&targeted oligonucleotide probes to identify small
phytoplankton by flow cytometry. !++l. En4i"on. 8i)"obiol. <%, 4;><&4;%7.
##. $imon, #., 8ampbell, 1., /rnolfsdottir, D., Croben, ,., Cuillou, 1., 1ange, M. and
Medlin, 1.). *4>>>+. /ligonucleotide probes for the identification of three algal
groups by dot blot and fluorescent whole&cell hybridization. 3. Eu/. 8i)"obiol ?A,
A<&@?.
#3. 1ange, M., Cuillou, 1., 2aulot, D., $imon, #., 'mann, ,.(., 1udwig, ". and
Medlin, 1.). *%FF<+. (dentification of the class 0rymnesiophyceae and the genus
*haeo)ystis with ribosomal ,#'&targeted nucleic acid probes detected by flow
cytometry. 3. *hy)ol. 74, @;@&@<@.
#4. Cuillou, 1., Moon&van&der&$taay, $.J., 8laustre, .., 0artensky, -. and 2aulot, D.
*%FFF+. Diversity and abundance of 5olidophyceae *.eterokonta+ in two oceanic
regions. !++l. En4i"on. 8i)"obiol. <;, ?;4@&?;7<.
#5. $imon, #., 5renner, G., Ddvardsen, 5. and Medlin, 1.). *%FFA+. The identification
of (h"yso)h"omulina and *"ymnesium species *.aptophyta, 0rymnesiophyceae+
using fluorescent or chemiluminescent oligonucleotide probes9 a means for
improving studies on to!ic algae. Eu". 3. *hy)ol. 74, 7F7&?>%.
#6. Miller, 0.D. and $cholin, 8.'. *%FF@+. (dentification and enumeration of cultures
and wild *seuo-0it-s)hia *5acillariophyceae+ using species&specific 1$= r,#'&
targeted fluorescent probes and filter&based whole cell hybridization. 3. *hy)ol.
7?, 7A%&7@4.
#7. Gohn, =., 8embella, '., .ummert, 8., DlbrKchter, M., Croben, ,. and Medlin,
1.). *4>>7+ Discrimination of the to!igenic dinoflagellate species !lexan"ium
tama"ense and !lexan"ium osten,elii in co&occurring natural populations from
$cottish coastal waters. Eu". 3. *hy)ol.7@4 4;&?>.
#8. Moon&van der $taay, $.J., van der $taay, C.".M., Cuillou, 1., 8laustre, .. and
2aulot, D. *4>>>+. 'bundance and diversity of 0rymnesiophyceae in picoplankton
communities from the equatorial 0acific /cean inferred from %@$ rD#'
sequences. :imnol. 2)eano%". ?;, F@&%>F.
F
Groben & Medlin In situ hybridisation of phytoplankton
#9. 'mann, ,., -uchs, 5.M. and 5ehrens, $. *4>>%+ The identification of
microorganisms by fluorescence in situ hybridisation. 8urr. /pin. 5iotechnol. %4,
47%&47<.
30. 0arsons, M. 1., $cholin, 8. '., Miller, 0. D., Doucette, C. G., 0owell, 8. 1.,
-ry!ell, C. '., Dortch, L. and $oniat, T. M. *%FFF+ *seuo-nit-s)hia species
*5acillariophyceae+ in 1ouisiana coastal waters9 Molecular probe field trials,
genetic variability, and domoic acid analyses. 3. *hy)ol. 7; *$uppl.+, %7<@&%7A@.
31. $cholin, 8.'., 5uck, ).,., 5ritschgi, T., 8angelosi, C. and 8havez, -.0. *%FF<+
(dentification of *seuo-nit-s)hia aust"alis *5acillariophyceae+ using r,#'&
targeted probes in whole cell and sandwich hybridization formats. *hy)olo%ia 7;,
%F>&%FA.
3#. $cholin, 8., Miller, 0., 5uck, )., 8havez, -., .arris, 0., .aydock, 0., .oward, G.
and 8angelosi, C. *%FFA+ Detection and quantification of *seuo-nit-s)hia
aust"alis in cultured and natural populations using 1$= r,#'&targeted probes.
:imnol. 2)eano%". ?4, %4<;&%4A4.
&rotocol
M'TD,('1$
Reagents5 4; ! $DT buffer9 7.A; M #a8l, 4; mM DDT', >.; M Tris.8l
*p. A.@+, filter&sterilize through >.4 Mm pore&size filters and
store at room temperature
$aline Dt/. fi!ative9 %>>B ethanol9 distilled water9 4; ! $DT
*4;9497+ *vv+, prepare fresh for every e!periment
.ybridization buffer9 ; ! $DT, >.%B *vv+ (CD0'1&8'<7> *or
#onidet&0?>+, NB *vv+ -ormamide, filter&sterilize through >.4
Mm pore&size filters, add 7> Mg ml
&%
0oly ' and store at room
temperature, the percentage of formamide *N+ in the buffer
depends on the probe requirements
%>
Groben & Medlin In situ hybridisation of phytoplankton
;>B Dimethylformamide *DM-+9 not mandatory3 dilute DM-
with de&ionized water, use glassware only as concentrated DM-
can dissolve plastic
8itifluorD'0(&Mi!9 8itifluor *8itifluor 1td, 8ambridge, =)+9
distilled water *49%+ *vv+ containing D'0( at a final
concentration of % Mg ml
&%
-1=/,D$8D(#&labelled oligonucleotide probes, stored at O
4>P8 in the dark
#ail varnish
E2uip6ent5 -iltration unit with vacuum pump
0olycarbonate -ilters *>.4 Mm or 7.> Mm pore size+
Moisture chamber *i.e., plastic bo! with its inside covered by
7MM "hatman paper that is soaked with hybridization buffer+
.ybridization oven
Microscopic slides and cover slips
-luorescence microscope equipped with =2 lamp and
appropriate filter sets for probe label and D'0(
%%
Groben & Medlin In situ hybridisation of phytoplankton
METH#!"
%i7ation o0 sa6ples
%. $et up filtration unit. =se polycarbonate filters with pore
sizes matching the e!pected cell sizes *>.4 Mm pore size for
picoplankton and bacteria, 7 Mm pore size for larger
phytoplankton+. The cells should be filtered onto the shiny
side of the filter.
4. 0our sample into filtration unit, use enough liquid to evenly
cover the whole filter.
7. -ilter sample through by using the lowest amount of
vacuum possible in which still a constant flow occurs to
prevent breakage of delicate cells
?. 8lose connecting piece to pump with parafilm so that no
liquid is dripping through the filter during incubation and
the filter does not run dry.
;. 'dd ;&%; ml of freshly prepared saline Dt/. fi!ative so
that the whole filter is covered and incubate at room
temperature for % h. The solution will normally show strong
precipitation, but this does not influence the fi!ation. -or
samples that contain species with strong autofluorescence
incubate for 4 h. 'fterwards remove the solution by vacuum
filtration.
<. 'dd ;&%; ml of hybridization buffer *without formamide+,
incubate at room temperature for ; min and filter buffer
through.
A. (f strong autofluorescence is e!pected, add ;>B DM- to
cover the filter and incubate at room temperature for one
hour. ,epeat step < afterwards.
@. 5riefly air dry the filter on "hatman paper. "rapped filters
can be stored at ?P8 for at least 7 weeks, or processed
immediately.
%4
Groben & Medlin In situ hybridisation of phytoplankton
Hybridi8ation
%. 8ut filter into pieces. ' filter of 4; mm diameter can easily
be cut into si! pieces if necessary, one of ?A mm diameter
into at least %4 pieces.
4. 0ut a filter piece onto a microscope slide, apply ;? Ml
hybridization buffer with < Ml probe *;> ng Ml
&%
stock+
directly onto the filter piece. Dnsure that the whole filter
piece is covered with liquid and if necessary use more
buffer&probe mi!ture. The fluorescent labelled probe is light
sensitive, so keep the filters in the dark for the rest of the
procedure, e.g., cover them during incubation times, and
e!pose them only shortly to light when handling them.
7. .ybridize slides at hybridization temperature *normally
;>P8 in a moisture chamber in the dark for % h+.
-ormamide *-'+ concentration for new probes need to be
determined empirically. .ybridize a filter piece each with >,
%>, 4>, 7>, ?> and ;>B -' in the buffer. 8hoose the highest
-' concentration for which target cells from defined lab
cultures give clear positive signals while non&target species
show no signal. ' moisture chamber can be made using
hybridization buffer without formamide.
?. "ash filter briefly in a small tray in % ! $DT buffer pre&
warmed to hybridization temperature and the dry the filter
on "hatman paper. 0ut the filter back onto a clean slide.
;. 'pply %>> Ml of pre&warmed %N $DT buffer onto the filter
and incubate again in the moisture chamber at the
hybridization temperature for ; min.
<. 5riefly dry the filter on "hatman paper, then put it back
onto a clean slide.
A. 'pply 4>&7> Ml of 8itifluorD'0( mi!ture directly onto the
filter, mount cover slip carefully so that the liquid covers
the filter piece without any air bubbles and seal the sides of
the cover slip with nail varnish.
%7
Groben & Medlin In situ hybridisation of phytoplankton
@. 2iew slides by fluorescence microscopy. 1ook at the
specimens using the filter for the D'0( stain and then
switch to the one for the fluorochrome, only target cells
should give positive, emerald green signals whereas non&
target cells show a brown&yellowish colour.
%?
Groben & Medlin In situ hybridisation of phytoplankton
%igure legend
-igure % a9 8ells of the dinoflagellate .ymnoinium mi/imotoi hybridized with a
dinoflagellate&specific -1=/,D$8D(#&labelled probe *left+ and same field seen with
D'0( staining *right+. 8ells were preserved with 0-' and $D$ detergent was used in
the hybridization buffer. The cells were ruptured after the hybridization and no signals
could be detected.
-igure % b9 8ells of the dinoflagellate .ymnoinium mi/imotoi hybridized with a
dinoflagellate&specific -1=/,D$8D(#&labelled probe *left+ and same field seen with
D'0( staining *right+. 8ells were preserved with saline&ethanol fi!ative and (CD0'1&
8'<7> detergent was used in the hybridization buffer. The cells were intact and gave
clear hybridization signals.
-igure % c9 8ells of the dinoflagellate !lexan"ium tama"ense hybridized with a
dinoflagellate&specific -1=/,D$8D(#&labelled probe probe *left+ and same field
seen with D'0( staining *right+. "ithout DM- treatment, cells show strong
autofluorescence that masks the probe signal and make them impossible to analyse.
-igure % d9 8ells of the dinoflagellate !lexan"ium tama"ense hybridized with a
dinoflagellate&specific -1=/,D$8D(#&labelled probe probe *left+ and same field
seen with D'0( staining *right+. 'fter DM- treatment, autofluorescence is mainly
reduced in nearly all cells and probe signals are clearly visible.
%;
DAPI stain